U.S. patent application number 17/286113 was filed with the patent office on 2021-12-09 for pumpless encapsulation of messenger rna.
The applicant listed for this patent is Translate Bio, Inc.. Invention is credited to Frank DeRosa, Michael Heartlein, Shrirang Karve, Priyal Patel, Natalia Vargas Montoya.
Application Number | 20210378962 17/286113 |
Document ID | / |
Family ID | 1000005837912 |
Filed Date | 2021-12-09 |
United States Patent
Application |
20210378962 |
Kind Code |
A1 |
Karve; Shrirang ; et
al. |
December 9, 2021 |
PUMPLESS ENCAPSULATION OF MESSENGER RNA
Abstract
The present invention provides, among other things, a process of
encapsulating messenger RNA (mRNA) in liposomes comprising a.
providing a first stream comprising an mRNA solution at a first
controlled flow rate, b. providing a second stream comprising a
lipid solution at a second controlled flow rate, and c. mixing the
first stream and the second stream to form mRNA-encapsulated
liposomes, wherein the first controlled flow rate and the second
controlled flow rate are achieved without use of a pump.
Inventors: |
Karve; Shrirang; (Lexington,
MA) ; Patel; Priyal; (Lexington, MA) ; Vargas
Montoya; Natalia; (Lexington, MA) ; DeRosa;
Frank; (Lexington, MA) ; Heartlein; Michael;
(Lexington, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Translate Bio, Inc. |
Lexington |
MA |
US |
|
|
Family ID: |
1000005837912 |
Appl. No.: |
17/286113 |
Filed: |
October 18, 2019 |
PCT Filed: |
October 18, 2019 |
PCT NO: |
PCT/US19/56936 |
371 Date: |
April 16, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62747838 |
Oct 19, 2018 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/7105 20130101;
A61K 9/1271 20130101; A61K 9/1277 20130101; A61K 48/00
20130101 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 31/7105 20060101 A61K031/7105 |
Claims
1. A process of encapsulating messenger RNA (mRNA) in liposomes
comprising a. providing a first stream comprising an mRNA solution
at a first controlled flow rate, b. providing a second stream
comprising a lipid solution at a second controlled flow rate, and
c. mixing the first stream and the second stream to form
mRNA-encapsulated liposomes, wherein the first controlled flow rate
and the second controlled flow rate are achieved without use of a
pump.
2. A process of encapsulating messenger RNA (mRNA) in liposomes
comprising a. providing a first stream comprising mRNA solution at
a first controlled flow rate. b. providing a second stream
comprising a lipid solution at a second controlled flow rate, and
c. mixing the first stream and the second stream to form
mRNA-encapsulated liposomes, d. wherein each of steps a-c is
performed under gravity feed and without external pressure.
3. The process of claim 1 or 2, wherein the first stream is
provided by a first conduit; and the second stream is provided by a
second conduit, and wherein the first conduit and the second
conduit are connected through a junction, thereby mixing the mRNA
solution and the lipid solution.
4. The process of claim 3, wherein the junction comprises a T
connector or a Y connector.
5. The process of claim 3 or 4, wherein the first conduit is
connected to a first reservoir containing the mRNA solution and the
second conduit is connected to a second reservoir containing the
lipid solution.
6. The process of any preceding claim, wherein a first constriction
controls the first controlled flow rate and a second constriction
controls the second controlled flow rate.
7. The process of claim 6, wherein the first constriction and
second constriction provide controlled flow rates that are the
same.
8. The process of claim 6, wherein the first constriction and the
second constriction provide controlled flow rates that are
different.
9. The process of claim 8, wherein the first controlled flow rate
to second control flow rate is at a ratio of about 1.2.times.,
1.5.times., 1.8.times., 2.0.times., 2.5.times., by 1.2.times. or
greater, 1.5.times. or greater, 1.8.times. or greater, 2.0.times.
or greater, 2.5.times. or greater.
10. The process of claim 8, wherein the second controlled flow rate
to first control flow rate is at a ratio of about 1.2.times.,
1.5.times., 1.8.times., 2.0.times., 2.5.times., by 1.2.times. or
greater, 1.5.times. or greater, 1.8.times. or greater, 2.0.times.
or greater, 2.5.times. or greater.
11. The process of claim 6, wherein the first constriction
comprises a first diameter of the first conduit and the second
constriction comprises a second diameter of the second conduit.
12. The process of claim 6, wherein the first constriction
comprises a first diameter of a first reservoir and the second
constriction comprises a second diameter of a second reservoir.
13. The process of claim 6, wherein the first constriction
comprises a first diameter of a first reservoir-conduit connection
and the second constriction comprises a second diameter of a second
reservoir-conduit connection.
14. The process of claim 6, wherein the first constriction
comprises a first diameter of a first conduit-junction connection
and the second constriction comprises a second diameter of a second
conduit-junction connection.
15. The process any one of claims 6-10, wherein the first
constriction comprises a first diameter of a first arm of a
junction and the second constriction comprises a second diameter of
a second arm of the junction.
16. The process of any of claims 6-15, wherein the first diameter
is identical to the second diameter.
17. The process of any one of claims 6-15, wherein the first
diameter is different from the second diameter.
18. The process of claim 17, wherein the first diameter is larger
than the second diameter.
19. The process of claim 18, wherein the first diameter is larger
than the second diameter by 1.2.times., 1.5.times., 1.8.times.,
2.0.times., 2.5.times., by 1.2.times. or greater, 1.5.times. or
greater, 1.8.times. or greater, 2.0.times. or greater, 2.5.times.
or greater.
20. The process of claim 18, wherein the first diameter is larger
than the second diameter in an amount that provides a first
controlled flow rate to second controlled flow rate ratio that is
1:1, 2:1, 3:1, 4:1, 5:1, 10:1, 1:1 or greater, 2:1 or greater, 3:1
or greater, or 4:1 or greater, or 5:1 or greater, or 10:1 or
greater.
21. The process of any one of claims 11-20, wherein the first
diameter of the first conduit is selected from the following
ranges: 0.1 mm-1 mm, 1 mm-100 mm, 100 mm-1 cm, 1 cm-100 cm.
22. The process of any one of claims 11-20, wherein the second
diameter of the second conduit is selected from the following
ranges: 0.1 mm-1 mm, 1 mm-100 mm, 100 mm-1 cm, 1 cm-100 cm.
23. The process of any one of claims 6-22, wherein the first
controlled flow rate ranges from about 0.1-1 mL/min, 1-150 mL/min,
150-250 mL/min, 250-500 mL/min, 500-1000 mL/min, 1000-2000 mL/min,
2000-3000 mL/min, 3000-4000 mL/min, or 4000-5000 mL/min.
24. The process of claim 23, wherein the first controlled flow rate
is about 200 mL/min.
25. The process of any one of claims 6-22, wherein the second
controlled flow rate ranges from about 0.1-1 mL/min, 1-150 mL/min,
150-250 mL/min, 250-500 mL/min, 500-1000 mL/min, 1000-2000 mL/min,
2000-3000 mL/min, 3000-4000 mL/min, or 4000-5000 mL/min.
26. The process of claim 25, wherein the second controlled flow
rate is about 50 mL/min.
27. The process of any one of the preceding claims, wherein the
lipid solution comprises one or more cationic lipids, one or more
helper lipids, and one or more PEG-modified lipids.
28. The process of claim 27, wherein the lipid solution further
comprises one or more cholesterol-based lipids.
29. The process of claim 28, wherein the one or more
cholesterol-based lipids are cholesterol and/or PEGylated
cholesterol.
30. The process of any one of the preceding claims, wherein the
lipid solution comprises pre-formed lipid nanoparticles.
31. The process of any one of the preceding claims, wherein the
lipid solution is a suspension of pre-formed lipid
nanoparticles.
32. The process of any one of the preceding claims, wherein the
first stream comprises about 50% water or greater and the second
stream comprises about 50% ethanol or greater.
33. The process of any one of the preceding claims, wherein the
first stream comprises about 85-99% water and the second stream
comprises about 85-99% ethanol.
34. The process of any one of claims 1-32, wherein each of the
first stream and the second stream comprises 50% water or
greater.
35. The process of any one of the preceding claims, wherein the
process results in lipid nanoparticles have a size ranging from
about 75-150 nm.
36. The process of any one of the preceding claims, wherein about
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99%, or 100% of the lipid nanoparticles have a size of 100 nm or
less.
37. The process of any one of the preceding claims, wherein greater
than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the
lipid nanoparticles have a size ranging from 50-80 nm.
38. The process of any one of the preceding claims, wherein the
process results in an encapsulation efficiency of at least about
70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.
39. The process of any one of the preceding claims, wherein the
process results in at least about 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% recovery of mRNA.
40. The process of any one of the preceding claims, wherein the
process results in at least 0.1 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 100
mg, 500 mg, or 1,000 mg of encapsulated mRNA.
41. The process of any one of the preceding claims, wherein the
process results in lipid nanoparticles that do not require further
purification.
42. The process of any one of the preceding claims, wherein the
process further comprises a step of collecting lipid nanoparticles
in a receptacle or conduit.
43. The process of any one of the preceding claims, wherein the
mRNA is codon-optimized.
44. The process of any one of the preceding claims, wherein the
mRNA is unmodified.
45. The process of any one of claims 1-43, wherein the mRNA is
modified.
46. The process of any one of the preceding claims, wherein the
process includes multiple pairs of first streams and corresponding
second streams.
47. The process of any one of the preceding claims, wherein in step
c the mixing of each of the pair of first and second streams occurs
simultaneously.
48. The process of claim 46, wherein the process comprises at least
10, 20, 30, 40, 50, 100, 150, 200 pairs of the first streams and
the second stream.
49. The process of claim 48, wherein each individual first stream
provides a different mRNA solution.
50. The process of claim 48, wherein at least a subset of first
streams provides a same mRNA solution.
51. The process of any one of claims 46-50, wherein each individual
second stream provides a different lipid solution.
52. The process of any one of claims 46-50, wherein at least a
subset of second streams provide a same lipid solution.
53. A method of delivering mRNA for in vivo protein production
comprising administering into a subject a composition of lipid
nanoparticles encapsulating mRNA generated by a process of any one
of the preceding claims.
54. A system for encapsulating messenger RNA (mRNA) in lipid
nanoparticles comprising a first conduit for providing an mRNA
solution at a first controlled flow rate, and a second conduit for
providing a lipid solution at a second controlled flow rate,
wherein the first conduit and the second conduit are connected
through a junction to facilitate mixing of the mRNA solution and
the lipid solution, and wherein the first controlled flow rate and
the second controlled flow rate are achieved without use of a
pump.
55. The system of claim 54, wherein the junction comprises a T
connector or a Y connector.
56. The system of claim 54 or 55, wherein the first conduit is
connected to a first reservoir for containing the mRNA solution and
the second conduit is connected to a second reservoir for
containing the lipid solution.
57. The system of any one of claims 54-56, wherein the first
conduit has a first diameter and the second conduit has a second
diameter.
58. The system of claim 57, wherein the first diameter is identical
to the second diameter.
59. The system of claim 57, wherein the first diameter is different
from the second diameter.
60. The system of claim 59, wherein the first dimeter is larger
than the second diameter.
61. The system of any one of claims 57-60, wherein the first
diameter of the first conduit is selected from the following
ranges: 0.1 mm-1 mm, 1 mm-100 mm, 100 mm-1 cm, 1 cm-100 cm.
62. The system of any one of claims 57-61, wherein the second
diameter of the second conduit is selected from the following
ranges: 0.1 mm-1 mm, 1 mm-100 mm, 100 mm-1 cm, 1 cm-100 cm.
63. The system of any one of claims 54-62, wherein the system
further comprises a receptacle or conduit to collect resulting
lipid nanoparticles.
64. The system of any one of claims 54-63, wherein the system
includes multiple pairs of first conduits and corresponding second
conduits.
65. The system of claim 64, wherein the system comprises at least
10, 20, 30, 40, 50, 100, 150, 200 pairs of the first conduit and
the second conduit.
66. The system of claim 65, wherein each of the first and second
conduits are connected to their respective first and second
reservoirs.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Provisional
Application Ser. No. 62/747,838 filed Oct. 19, 2018, the
disclosures of which are hereby incorporated by reference.
BACKGROUND
[0002] Messenger RNA therapy (MRT) is becoming an increasingly
important approach for the treatment of a variety of diseases. MRT
involves administration of messenger RNA (mRNA) into a patient in
need thereof. The administered mRNA produces a protein encoded by
the mRNA within the patient's body. Liposomes and/or lipid
nanoparticles are commonly used to encapsulate mRNA for efficient
in vivo delivery of mRNA. However, current methods for producing
mRNA-loaded lipid nanoparticles suffer from poor encapsulation
efficiency, low mRNA recovery and/or heterogeneous particle sizes.
Another disadvantage to current methods is the need to use
specialized equipment, such as gear pumps, peristaltic pumps and
the like for the encapsulation process. Such specialized equipment
may not be readily available in certain locations, such as
patient-care settings.
SUMMARY OF INVENTION
[0003] The present invention provides, among other things, an
improved process for lipid nanoparticle formulation and mRNA
encapsulation. In particular, the present invention is based on the
surprising discovery that a gravity-based mixing process for lipid
encapsulation of nucleic acids allows for reproducible, highly
efficient encapsulation efficiencies.
[0004] The present invention provides a cost effective, robust and
user friendly mRNA encapsulation method that uses a gravity-based
mixing process. Some of the benefits of a gravity-based mixing
process for the encapsulation of nucleic acids include, for
example, less variability, a pulse-less flow of liquid allowing for
precise, reproducible mixing flow rates without the need of
machine-driven actuators. Other advantages of the gravity mixing
process described herein include no calibrations or lengthy setups,
cost efficiency, low maintenance and robust physical design (e.g.
no moving parts and/or minimum accessories). The invention
described herein provides, among other things, a scalable system
which allows for multiple formulations that could be made at the
same time independent of capacity and with minimal losses of
material. Furthermore, the invention described herein provides for
flexibility of process variables at least because the flow under
gravity can be fine-tuned based on diameter of the flow path, which
in turn allows for more flexibility with respect to flow rates.
[0005] Thus, in one aspect, the present invention provides a
process of encapsulating messenger RNA (mRNA) in liposomes
comprising a. providing a first stream comprising an mRNA solution
at a first controlled flow rate, b. providing a second stream
comprising a lipid solution at a second controlled flow rate, and
c. mixing the first stream and the second stream to form
mRNA-encapsulated liposomes, wherein the first controlled flow rate
and the second controlled flow rate are achieved without use of a
pump.
[0006] In another aspect, the present invention provides a process
of encapsulating messenger RNA (mRNA) in liposomes comprising a.
providing a first stream comprising mRNA solution at a first
controlled flow rate, b. providing a second stream comprising a
lipid solution at a second controlled flow rate, and c. mixing the
first stream and the second stream to form mRNA-encapsulated
liposomes, d. wherein each of steps a-c is performed under gravity
feed and without external pressure.
[0007] In some embodiments, the process of claim 1 or 2, wherein
the first stream is provided by a first conduit; and the second
stream is provided by a second conduit, and wherein the first
conduit and the second conduit are connected through a junction,
thereby mixing the mRNA solution and the lipid solution.
[0008] In some embodiments, the junction comprises a T connector or
a Y connector.
[0009] In some embodiments, the first conduit is connected to a
first reservoir containing the mRNA solution and the second conduit
is connected to a second reservoir containing the lipid
solution.
[0010] In some embodiments, a first constriction controls the first
controlled flow rate and a second constriction controls the second
controlled flow rate.
[0011] In some embodiments, the first constriction and the second
constriction provide controlled flow rates that are the same.
[0012] In some embodiments, the first constriction and the second
constriction provide controlled flow rates that are different.
[0013] In some embodiments, the first controlled flow rate to
second control flow rate is at a ratio of about 1.0.times.. In some
embodiments, the first controlled flow rate to second control flow
rate is at a ratio of about 1.2.times.. In some embodiments, the
first controlled flow rate to second control flow rate is at a
ratio of about 1.5.times.. In some embodiments, the first
controlled flow rate to second control flow rate is at a ratio of
about 1.8.times.. In some embodiments, the first controlled flow
rate to second control flow rate is at a ratio of about 2.0.times..
In some embodiments, the first controlled flow rate to second
control flow rate is at a ratio of about 2.5.times.. In some
embodiments, the first controlled flow rate to second control flow
rate is at a ratio of about 3.0.times.. In some embodiments, the
first controlled flow rate to second control flow rate is at a
ratio of about 1.0.times. or greater. In some embodiments, the
first controlled flow rate to second control flow rate is at a
ratio of about 1.2.times. or greater. In some embodiments, the
first controlled flow rate to second control flow rate is at a
ratio of about 1.5.times. or greater. In some embodiments, the
first controlled flow rate to second control flow rate is at a
ratio of about 1.8.times. or greater. In some embodiments, the
first controlled flow rate to second control flow rate is at a
ratio of about 2.0.times. or greater. In some embodiments, the
first controlled flow rate to second control flow rate is at a
ratio of about 2.5.times. or greater. In some embodiments, the
first controlled flow rate to second control flow rate is at a
ratio of about 3.0.times. or greater. In some embodiments, the
first controlled flow rate to second control flow rate is at a
ratio of about 3.5.times. or greater. In some embodiments, the
first controlled flow rate to second control flow rate is at a
ratio of about 4.0.times. or greater.
[0014] In some embodiments, the second controlled flow rate to
first control flow rate is at a ratio of about 1.0.times.. In some
embodiments, the second controlled flow rate to first control flow
rate is at a ratio of about 1.2.times.. In some embodiments, the
second controlled flow rate to first control flow rate is at a
ratio of about 1.5.times.. In some embodiments, the second
controlled flow rate to first control flow rate is at a ratio of
about 1.8.times.. In some embodiments, the second controlled flow
rate to first control flow rate is at a ratio of about 2.0.times..
In some embodiments, the second controlled flow rate to first
control flow rate is at a ratio of about 2.5.times.. In some
embodiments, the second controlled flow rate to first control flow
rate is at a ratio of about 3.0.times.. In some embodiments, the
second controlled flow rate to first control flow rate is at a
ratio of about 1.0.times. or greater. In some embodiments, the
second controlled flow rate to first control flow rate is at a
ratio of about 1.2.times. or greater. In some embodiments, the
second controlled flow rate to first control flow rate is at a
ratio of about 1.5.times. or greater. In some embodiments, the
second controlled flow rate to first control flow rate is at a
ratio of about 1.8.times. or greater. In some embodiments, the
second controlled flow rate to first control flow rate is at a
ratio of about 2.0.times. or greater. In some embodiments, the
second controlled flow rate to first control flow rate is at a
ratio of about 2.5.times. or greater. In some embodiments, the
second controlled flow rate to first control flow rate is at a
ratio of about 3.0.times. or greater. In some embodiments, the
second controlled flow rate to first control flow rate is at a
ratio of about 3.5.times. or greater. In some embodiments, the
second controlled flow rate to first control flow rate is at a
ratio of about 4.0.times. or greater.
[0015] In some embodiments, the first constriction comprises a
first diameter of the first conduit and the second constriction
comprises a second diameter of the second conduit.
[0016] In some embodiments, the first constriction comprises a
first diameter of a first reservoir and the second constriction
comprises a second diameter of a second reservoir.
[0017] In some embodiments, the first constriction comprises a
first diameter of a first reservoir-conduit connection and the
second constriction comprises a second diameter of a second
reservoir-conduit connection.
[0018] In some embodiments, the first constriction comprises a
first diameter of a first conduit-junction connection and the
second constriction comprises a second diameter of a second
conduit-junction connection. The process of any of claims 7-10,
wherein the first diameter is identical to the second diameter.
[0019] In some embodiments, the first diameter is different from
the second diameter.
[0020] In some embodiments, the first diameter is larger than the
second diameter.
[0021] In some embodiments, the first diameter is larger than the
second diameter by 1.2.times., 1.5.times., 1.8.times., 2.0.times.,
2.5.times., by 1.2.times. or greater, 1.5.times. or greater,
1.8.times. or greater, 2.0.times. or greater, 2.5.times. or
greater. In some embodiments, the first diameter is larger than the
second diameter by about 3.0.times., 3.5.times., 4.0.times.,
4.5.times., 5.0.times., 5.5.times., 6.0 or greater.
[0022] In some embodiments, the first diameter is larger than the
second diameter in an amount that provides a first controlled flow
rate to second controlled flow rate ratio that is 1:1, 2:1, 3:1,
4:1, 5:1, 10:1, 1:1 or greater, 2:1 or greater, 3:1 or greater, or
4:1 or greater, or 5:1 or greater, or 10:1 or greater.
[0023] In some embodiments, the first diameter of the first conduit
is selected from the following ranges: 0.1 mm-1 mm, 1 mm-100 mm,
100 mm-1 cm, 1 cm-100 cm.
[0024] In some embodiments, the second diameter of the second
conduit is selected from the following ranges: 0.1 mm-1 mm, 1
mm-100 mm, 100 mm-1 cm, 1 cm-100 cm.
[0025] In some embodiments, the first controlled flow rate ranges
from about 0.1-1 mL/min, 1-150 mL/min, 150-250 mL/min, 250-500
mL/min, 500-1000 mL/min, 1000-2000 mL/min, 2000-3000 mL/min,
3000-4000 mL/min, or 4000-5000 mL/min.
[0026] In some embodiments, the first controlled flow rate is about
50 mL/min. In some embodiments, the first controlled flow rate is
about 100 mL/min. In some embodiments, the first controlled flow
rate is about 150 mL/min. In some embodiments, the first controlled
flow rate is about 200 mL/min. In some embodiments, the first
controlled flow rate is about 250 mL/min. In some embodiments, the
first controlled flow rate is about 300 mL/min. In some
embodiments, the first controlled flow rate is about 400 mL/min. In
some embodiments, the first controlled flow rate is about 500
mL/min.
[0027] In some embodiments, the second controlled flow rate ranges
from about 0.1-1 mL/min, 1-150 mL/min, 150-250 mL/min, 250-500
mL/min, 500-1000 mL/min, 1000-2000 mL/min, 2000-3000 mL/min,
3000-4000 mL/min, or 4000-5000 mL/min.
[0028] In some embodiments, the second controlled flow rate is
about 10 mL/min. In some embodiments, the second controlled flow
rate is about 50 mL/min. In some embodiments, the second controlled
flow rate is about 100 mL/min. In some embodiments, the second
controlled flow rate is about 150 mL/min. In some embodiments, the
second controlled flow rate is about 200 mL/min. In some
embodiments, the second controlled flow rate is about 250 mL/min.
In some embodiments, the second controlled flow rate is about 300
mL/min. In some embodiments, the second controlled flow rate is
about 400 mL/min. In some embodiments, the second controlled flow
rate is about 500 mL/min.
[0029] In some embodiments, the lipid solution comprises one or
more cationic lipids, one or more helper lipids, and one or more
PEG-modified lipids.
[0030] In some embodiments, the lipid solution further comprises
one or more cholesterol-based lipids.
[0031] In some embodiments, the one or more cholesterol-based
lipids are cholesterol and/or PEGylated cholesterol.
[0032] In some embodiments, the lipid solution comprises pre-formed
lipid nanoparticles.
[0033] In some embodiments, the lipid solution is a suspension of
pre-formed lipid nanoparticles.
[0034] In some embodiments, the first stream comprises about 50%
water or greater and the second stream comprises about 50% ethanol
or greater.
[0035] In some embodiments, the first stream comprises about 85-99%
water and the second stream comprises about 85-99% ethanol.
[0036] In some embodiments, each of the first stream and the second
stream comprises 50% water or greater.
[0037] In some embodiments, the process results in lipid
nanoparticles have a size ranging from about 40-150 nm.
[0038] In some embodiments, about 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the lipid
nanoparticles have a size of 150 nm or less.
[0039] In some embodiments, about 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the lipid
nanoparticles have a size of 100 nm or less.
[0040] In some embodiments, greater than about 70%, 75%, 80%, 85%,
90%, 95%, 96%, 97%, 98%, 99% of the lipid nanoparticles have a size
ranging from 50-80 nm.
[0041] In some embodiments, the process results in an encapsulation
efficiency of at least about 60%. In some embodiments, the process
results in an encapsulation efficiency of at least about 65%. In
some embodiments, the process results in an encapsulation
efficiency of at least about 70%. In some embodiments, the process
results in an encapsulation efficiency of at least about 75%. In
some embodiments, the process results in an encapsulation
efficiency of at least about 80%. In some embodiments, the process
results in an encapsulation efficiency of at least about 85%. In
some embodiments, the process results in an encapsulation
efficiency of at least about 90%. In some embodiments, the process
results in an encapsulation efficiency of at least about 95%. In
some embodiments, the process results in an encapsulation
efficiency of at least about 96%. In some embodiments, the process
results in an encapsulation efficiency of at least about 97%. In
some embodiments, the process results in an encapsulation
efficiency of at least about 98%. In some embodiments, the process
results in an encapsulation efficiency of at least about 99%.
[0042] In some embodiments, the process results in at least about
50% recovery of mRNA. In some embodiments, the process results in
at least about 55% recovery of mRNA. In some embodiments, the
process results in at least about 60% recovery of mRNA. In some
embodiments, the process results in at least about 65% recovery of
mRNA. In some embodiments, the process results in at least about
70% recovery of mRNA. In some embodiments, the process results in
at least about 75% recovery of mRNA. In some embodiments, the
process results in at least about 80% recovery of mRNA. In some
embodiments, the process results in at least about 85% recovery of
mRNA. In some embodiments, the process results in at least about
90% recovery of mRNA. In some embodiments, the process results in
at least about 95% recovery of mRNA. In some embodiments, the
process results in at least about 96% recovery of mRNA. In some
embodiments, the process results in at least about 97% recovery of
mRNA. In some embodiments, the process results in at least about
98% recovery of mRNA. In some embodiments, the process results in
at least about 99% recovery of mRNA.
[0043] In some embodiments, the process results in at least 0.1 mg
of encapsulated mRNA. In some embodiments, the process results in
at least 0.5 mg of encapsulated mRNA. In some embodiments, the
process results in at least 1 mg of encapsulated mRNA. In some
embodiments, the process results in at least 5 mg of encapsulated
mRNA. In some embodiments, the process results in at least 10 mg of
encapsulated mRNA. In some embodiments, the process results in at
least 100 mg of encapsulated mRNA. In some embodiments, the process
results in at least 500 mg of encapsulated mRNA. In some
embodiments, the process results in at least 1,000 mg of
encapsulated mRNA.
[0044] In some embodiments, the process results in lipid
nanoparticles that do not require further purification.
[0045] In some embodiments, the process further comprises a step of
collecting lipid nanoparticles in a receptacle or conduit.
[0046] In some embodiments, the mRNA is codon-optimized.
[0047] In some embodiments, the mRNA is unmodified.
[0048] In some embodiments, the mRNA is modified.
[0049] In some embodiments, the process includes multiple pairs of
first streams and corresponding second streams.
[0050] In some embodiments, in step c the mixing of each of the
pair of first and second streams occurs simultaneously.
[0051] In some embodiments, the process comprises at least 10, 20,
30, 40, 50, 100, 150, 200 pairs of the first streams and the second
stream.
[0052] In some embodiments, each individual first stream provides a
different mRNA
Solution
[0053] In some embodiments, at least a subset of first streams
provides a same mRNA
Solution
[0054] In some embodiments, each individual second stream provides
a different lipid solution.
[0055] In some embodiments, at least a subset of second streams
provide a same lipid solution.
[0056] In another aspect, the present invention provides a method
of delivering mRNA for in vivo protein production comprising
administering into a subject a composition of lipid nanoparticles
encapsulating mRNA generated by a process of any one of the
preceding claims.
[0057] In another aspect, the present invention provides a system
for encapsulating messenger RNA (mRNA) in lipid nanoparticles
comprising a first conduit for providing an mRNA solution at a
first controlled flow rate, and a second conduit for providing a
lipid solution at a second controlled flow rate, wherein the first
conduit and the second conduit are connected through a junction to
facilitate mixing of the mRNA solution and the lipid solution, and
wherein the first controlled flow rate and the second controlled
flow rate are achieved without use of a pump.
[0058] In some embodiments, the junction comprises a T connector or
a Y connector.
[0059] In some embodiments, the first conduit is connected to a
first reservoir for containing the mRNA solution and the second
conduit is connected to a second reservoir for containing the lipid
solution.
[0060] In some embodiments, the first conduit has a first diameter
and the second conduit has a second diameter.
[0061] In some embodiments, the first diameter is identical to the
second diameter.
[0062] In some embodiments, the first diameter is different from
the second diameter.
[0063] In some embodiments, the first dimeter is larger than the
second diameter.
[0064] In some embodiments, the first diameter of the first conduit
is selected from the following ranges: 0.1 mm-1 mm, 1 mm-100 mm,
100 mm-1 cm, 1 cm-100 cm.
[0065] In some embodiments, the second diameter of the second
conduit is selected from the following ranges: 0.1 mm-1 mm, 1
mm-100 mm, 100 mm-1 cm, 1 cm-100 cm.
[0066] In some embodiments, the system further comprises a
receptacle or conduit to collect resulting lipid nanoparticles.
[0067] Other features, objects, and advantages of the present
invention are apparent in the detailed description, drawings and
claims that follow. It should be understood, however, that the
detailed description, the drawings, and the claims, while
indicating embodiments of the present invention, are given by way
of illustration only, and should not be construed as being
limiting. Various changes and modifications within the scope of the
invention will become apparent to those skilled in the art.
BRIEF DESCRIPTION OF THE DRAWINGS
[0068] The drawings are for illustration purposes only and not for
limitation.
[0069] FIG. 1 is a schematic of a pumpless nucleic acid lipid
nanoparticle encapsulation process. The schematic depicts Reservoir
1 which houses a nucleic acid solution (mRNA), Reservoir 2 which
houses lipids or empty lipid nanoparticles (LNPs), Conduits 1 and
Conduit 2 though which liquid from Reservoirs 1 and 2 flow,
respectively. The flow from Conduit 1 (Flow 1) and from Conduit 2
(Flow 2) meet and mix at the junction (depicted as a T-junction).
Upon mixing, the encapsulated nucleic acid is collected in a
receptacle.
[0070] FIGS. 2A and 2B are schematics that depict an exemplary
process comprising a "T" junction (FIG. 2A) or a "Y" junction (FIG.
2B) at the point of mixture.
[0071] FIGS. 3A and 3B are schematics that depict the influence of
diameter on the flow of liquid through the process conduits. FIG.
3A depicts a conduit that has a large diameter. FIG. 3B depicts a
conduit that has a small diameter.
[0072] FIG. 4 (panels A-D) is a series of schematics that show
exemplary locations to place constrictions to regulate the diameter
and flow of liquid in the process. FIG. 4, panel A depicts a
Reservoir, conduit and junction that does not have a constrictor.
FIG. 4, panel B depicts a reservoir and a conduit, in which the
conduit has a constrictor attached. FIG. 4, panel C depicts a
reservoir and a conduit in which the conduit has a constrictor
attached near the junction. FIG. 4, panel D depicts a reservoir and
a conduit in which a constrictor is placed over the reservoir.
[0073] FIGS. 5A and 5B is a series of schematics that depict one
embodiment used to control the flow rate and the resultant mixing
in the process. FIG. 5A shows a reservoir, conduit and junction, in
which the junction is in an upwards position. This upwards position
prevents flow of liquid and allows the lines to purge. FIG. 5B
depicts a reservoir, conduit and junction, in which the junction is
in an extended position. This downward position allows the conduits
to fill with liquid and be mixed at the junction.
[0074] FIG. 6 depicts a schematic of the process in fixed system in
which liquids in reservoirs 1 and 2 follow the flow of gravity,
through the conduit, and mix at the junction.
[0075] FIG. 7 depicts a schematic of a tandem process in which
liquids are added to pairs of reservoirs at the same time, followed
by the addition of liquids the next pairs of reservoirs in
succession
[0076] FIG. 8 depicts a schematic of a high throughput process in
which multiple pairs of conduits are used.
DEFINITIONS
[0077] In order for the present invention to be more readily
understood, certain terms are first defined below. Additional
definitions for the following terms and other terms are set forth
throughout the specification.
[0078] Alkyl: As used herein, "alkyl" refers to a radical of a
straight-chain or branched saturated hydrocarbon group having from
1 to 20 carbon atoms ("C.sub.1-20 alkyl"). In some embodiments, an
alkyl group has 1 to 3 carbon atoms ("C.sub.1-3 alkyl"). Examples
of C.sub.1-3 alkyl groups include methyl (C.sub.1), ethyl
(C.sub.2), n-propyl (C.sub.3), and isopropyl (C.sub.3). In some
embodiments, an alkyl group has 8 to 12 carbon atoms ("C.sub.8-12
alkyl"). Examples of C.sub.8-12 alkyl groups include, without
limitation, n-octyl (C.sub.8), n-nonyl (C.sub.9), n-decyl
(C.sub.10), n-undecyl (C.sub.11), n-dodecyl (C.sub.12) and the
like. The prefix "n-" (normal) refers to unbranched alkyl groups.
For example, n-C.sub.8 alkyl refers to --(CH.sub.2).sub.7CH.sub.3,
n-C.sub.10 alkyl refers to --(CH.sub.2).sub.9CH.sub.3, etc.
[0079] Amino acid: As used herein, term "amino acid," in its
broadest sense, refers to any compound and/or substance that can be
incorporated into a polypeptide chain. In some embodiments, an
amino acid has the general structure H.sub.2N--C(H)(R)--COOH. In
some embodiments, an amino acid is a naturally occurring amino
acid. In some embodiments, an amino acid is a synthetic amino acid;
in some embodiments, an amino acid is a d-amino acid; in some
embodiments, an amino acid is an 1-amino acid. "Standard amino
acid" refers to any of the twenty standard 1-amino acids commonly
found in naturally occurring peptides. "Nonstandard amino acid"
refers to any amino acid, other than the standard amino acids,
regardless of whether it is prepared synthetically or obtained from
a natural source. As used herein, "synthetic amino acid"
encompasses chemically modified amino acids, including but not
limited to salts, amino acid derivatives (such as amides), and/or
substitutions. Amino acids, including carboxy- and/or
amino-terminal amino acids in peptides, can be modified by
methylation, amidation, acetylation, protecting groups, and/or
substitution with other chemical groups that can change the
peptide's circulating half-life without adversely affecting their
activity. Amino acids may participate in a disulfide bond. Amino
acids may comprise one or posttranslational modifications, such as
association with one or more chemical entities (e.g., methyl
groups, acetate groups, acetyl groups, phosphate groups, formyl
moieties, isoprenoid groups, sulfate groups, polyethylene glycol
moieties, lipid moieties, carbohydrate moieties, biotin moieties,
etc.). The term "amino acid" is used interchangeably with "amino
acid residue," and may refer to a free amino acid and/or to an
amino acid residue of a peptide. It will be apparent from the
context in which the term is used whether it refers to a free amino
acid or a residue of a peptide.
[0080] Atmospheric pressure: As used herein, the term "atmospheric
pressure" means the pressure exerted by the weight of the
atmosphere, which at sea level has a mean value of about 101,325
pascals.
[0081] Approximately or about: As used herein, the term
"approximately" or "about," as applied to one or more values of
interest, refers to a value that is similar to a stated reference
value. In certain embodiments, the term "approximately" or "about"
refers to a range of values that fall within 25%, 20%, 19%, 18%,
17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,
2%, 1%, or less in either direction (greater than or less than) of
the stated reference value unless otherwise stated or otherwise
evident from the context (except where such number would exceed
100% of a possible value).
[0082] Encapsulation: As used herein, the term "encapsulation," or
grammatical equivalent, refers to the process of confining an
individual mRNA molecule within a nanoparticle.
[0083] Gravity Feed: As used herein, the term "gravity feed" means
using gravity to move a substance (e.g. a liquid) from one place to
another without the use of a pump.
[0084] Improve, increase, or reduce: As used herein, the terms
"improve," "increase" or "reduce," or grammatical equivalents,
indicate values that are relative to a baseline measurement, such
as a measurement in the same individual prior to initiation of the
treatment described herein, or a measurement in a control subject
(or multiple control subject) in the absence of the treatment
described herein. A "control subject" is a subject afflicted with
the same form of disease as the subject being treated, who is about
the same age as the subject being treated.
[0085] Impurities: As used herein, the term "impurities" refers to
substances inside a confined amount of liquid, gas, or solid, which
differ from the chemical composition of the target material or
compound. Impurities are also referred to as contaminants.
[0086] In Vitro: As used herein, the term "in vitro" refers to
events that occur in an artificial environment, e.g., in a test
tube or reaction vessel, in cell culture, etc., rather than within
a multi-cellular organism.
[0087] In Vivo: As used herein, the term "in vivo" refers to events
that occur within a multi-cellular organism, such as a human and a
non-human animal. In the context of cell-based systems, the term
may be used to refer to events that occur within a living cell (as
opposed to, for example, in vitro systems).
[0088] Isolated: As used herein, the term "isolated" refers to a
substance and/or entity that has been (1) separated from at least
some of the components with which it was associated when initially
produced (whether in nature and/or in an experimental setting),
and/or (2) produced, prepared, and/or manufactured by the hand of
man. Isolated substances and/or entities may be separated from
about 10%, about 20%, about 30%, about 40%, about 50%, about 60%,
about 70%, about 80%, about 90%, about 91%, about 92%, about 93%,
about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,
or more than about 99% of the other components with which they were
initially associated. In some embodiments, isolated agents are
about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,
about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,
or more than about 99% pure. As used herein, a substance is "pure"
if it is substantially free of other components. As used herein,
calculation of percent purity of isolated substances and/or
entities should not include excipients (e.g., buffer, solvent,
water, etc.).
[0089] messenger RNA (mRNA): As used herein, the term "messenger
RNA (mRNA)" refers to a polynucleotide that encodes at least one
peptide, polypeptide or protein. mRNA as used herein encompasses
both modified and unmodified RNA. mRNA may contain one or more
coding and non-coding regions. mRNA can be purified from natural
sources, produced using recombinant expression systems and
optionally purified, chemically synthesized, etc. Where
appropriate, e.g., in the case of chemically synthesized molecules,
mRNA can comprise nucleoside analogs such as analogs having
chemically modified bases or sugars, backbone modifications,
etc.
[0090] Nucleic acid: As used herein, the term "nucleic acid," in
its broadest sense, refers to any compound and/or substance that is
or can be incorporated into a polynucleotide chain. In some
embodiments, a nucleic acid is a compound and/or substance that is
or can be incorporated into a polynucleotide chain via a
phosphodiester linkage. In some embodiments, "nucleic acid" refers
to individual nucleic acid residues (e.g., nucleotides and/or
nucleosides). In some embodiments, "nucleic acid" refers to a
polynucleotide chain comprising individual nucleic acid residues.
In some embodiments, "nucleic acid" encompasses RNA as well as
single and/or double-stranded DNA and/or cDNA. Furthermore, the
terms "nucleic acid," "DNA," "RNA," and/or similar terms include
nucleic acid analogs, i.e., analogs having other than a
phosphodiester backbone. For example, the so-called "peptide
nucleic acids," which are known in the art and have peptide bonds
instead of phosphodiester bonds in the backbone, are considered
within the scope of the present invention. The term "nucleotide
sequence encoding an amino acid sequence" includes all nucleotide
sequences that are degenerate versions of each other and/or encode
the same amino acid sequence. Nucleotide sequences that encode
proteins and/or RNA may include introns. Nucleic acids can be
purified from natural sources, produced using recombinant
expression systems and optionally purified, chemically synthesized,
etc. Where appropriate, e.g., in the case of chemically synthesized
molecules, nucleic acids can comprise nucleoside analogs such as
analogs having chemically modified bases or sugars, backbone
modifications, etc. A nucleic acid sequence is presented in the 5'
to 3' direction unless otherwise indicated. In some embodiments, a
nucleic acid is or comprises natural nucleosides (e.g., adenosine,
thymidine, guanosine, cytidine, uridine, deoxyadenosine,
deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside
analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine,
pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5
propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine,
C.sub.5-bromouridine, C.sub.5-fluorouridine, C5-iodouridine,
C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine,
2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine,
8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and
2-thiocytidine); chemically modified bases; biologically modified
bases (e.g., methylated bases); intercalated bases; modified sugars
(e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and
hexose); and/or modified phosphate groups (e.g., phosphorothioates
and 5'-N-phosphoramidite linkages). In some embodiments, the
present invention is specifically directed to "unmodified nucleic
acids," meaning nucleic acids (e.g., polynucleotides and residues,
including nucleotides and/or nucleosides) that have not been
chemically modified in order to facilitate or achieve delivery.
[0091] Pump: As used herein, the term "pump" refers to a device for
driving or compressing fluids or gases. Multiple kinds of "pumps"
are known, and include for example diaphragm pumps, centrifugal
pumps, piston pumps, peristaltic pumps, pulse pumps and lobe pumps.
Pumps may include mechanically actuated devices and/or manually
actuated devices.
[0092] Salt: As used herein, the term "salt" refers to an ionic
compound that does or may result from a neutralization reaction
between an acid and a base.
[0093] Substantially: As used herein, the term "substantially"
refers to the qualitative condition of exhibiting total or
near-total extent or degree of a characteristic or property of
interest. One of ordinary skill in the biological arts will
understand that biological and chemical phenomena rarely, if ever,
go to completion and/or proceed to completeness or achieve or avoid
an absolute result. The term "substantially" is therefore used
herein to capture the potential lack of completeness inherent in
many biological and chemical phenomena.
[0094] Yield: As used herein, the term "yield" refers to the
percentage of mRNA recovered after encapsulation as compared to the
total mRNA as starting material. In some embodiments, the term
"recovery" is used interchangeably with the term "yield".
[0095] Various aspects of the invention are described in detail in
the following sections. The use of sections is not meant to limit
the invention. Each section can apply to any aspect of the
invention. In this application, the use of "or" means "and/or"
unless stated otherwise.
DETAILED DESCRIPTION
[0096] The present invention provides, among other things, an
improved process for lipid nanoparticle formulation and nucleic
acid encapsulation. The present invention is, in part, based on the
surprising discovery that nucleic acid encapsulation can be
reproducibly performed at atmospheric pressure using gravity to
control lipid and nucleic acid mixing rates to produce encapsulated
nucleic acids. Using gravity in this way, the mixing can be
performed without use of pumps or external pressure (e.g. pump
pressure), other than atmospheric pressure. Accordingly, the
pressures that are used to control the flow rate of the process
described herein are atmospheric pressure and head pressure (e.g.
pressure in a tube and reservoir). The encapsulation process
provided herein ensures stability of nucleic acids, including for
example mRNA, and results in high encapsulation efficiencies (e.g.
about between 75 and 95% or more encapsulation efficiencies).
[0097] The encapsulation process described herein allows for use of
the process in various settings, for example in large-scale
commercial process manufacturing where large volumes of pressurized
liquids such as ethanol may pose safety and handling concerns.
Other uses of the encapsulation process disclosed herein include
"bedside" commercial process manufacturing, for example, in the
preparation of small-scale commercial products on site such as in a
hospital pharmacy. In yet another use, the process described herein
can be used in a commercial process manufacturing setting in
locations where expensive equipment and maintenance may be
challenging. In yet another example of use, the process described
herein can be used in the high-throughput preparation of
encapsulated nucleic acids, for example for screening purposes,
where purchase, calibration and maintenance of large numbers of
pumps and other machinery inhibit quick and high-throughput
testing.
[0098] The encapsulation process described herein can be applied to
various other methods of encapsulating mRNA in lipid nanoparticles
described in the art. As used herein, Process A refers to a
conventional method of encapsulating mRNA by mixing mRNA with a
mixture of lipids, without first pre-forming the lipids into lipid
nanoparticles. As used herein, Process B refers to a process of
encapsulating messenger RNA (mRNA) by mixing pre-formed lipid
nanoparticles with mRNA. As compared to Process B, Process A does
not involve pre-formation of lipid nanoparticles. Process A and
Process B include those described in WO2016004318 and WO2018089801,
respectively, which are hereby incorporated by reference. In some
embodiments, the encapsulation process described herein can be used
to make empty lipid nanoparticles.
[0099] Accordingly, the invention provided herein allows for highly
efficient, reproducible lipid encapsulation of nucleic acids at
various scales and settings by the use of a gravity-based mixing
process to produce encapsulated nucleic acids.
Gravity-Based Encapsulation of Nucleic Acids
[0100] In one aspect of the disclosure, a process of encapsulating
a nucleic acid in liposomes is provided. In some embodiments, the
encapsulation process includes 1) a first reservoir to provide a
desired nucleic acid in aqueous solution; 2) a second reservoir to
provide a solution of lipids and/or lipid nanoparticles (LNPs); 3)
conduits for the first and second reservoirs to allow for flow of
nucleic acids, and lipids and/or LNPs; 4) a junction for mixing the
nucleic acids and lipids and/or LNPs; and 5) a receptacle or
conduit for collecting the mixed/encapsulated nucleic acids in
LNPs. A schematic of an exemplary process of encapsulating a
nucleic acid is shown in FIG. 1.
[0101] In some embodiments, the encapsulation process includes 1) a
first reservoir to provide an mRNA solution; 2) a second reservoir
to provide a solution of lipids and/or lipid nanoparticles (LNPs);
3) conduits for the first and the second reservoirs to allow for
flow of the mRNA solution, and the lipids and/or LNPs; 4) a
junction for mixing the mRNA and lipids and/or LNPs; and 5) a
receptacle or conduit for collecting the mixed the mixed
encapsulated mRNA in LNPs.
[0102] In some embodiments, the encapsulated mRNA is suitable to
deliver for in vivo protein production comprising administering
into a subject a composition of lipid nanoparticles encapsulating
mRNA generated by the process described herein. Accordingly, in
some embodiments, a method is provided of delivering mRNA for in
vivo protein production comprising administering into a subject a
composition of lipid nanoparticles encapsulating mRNA generated by
the process described herein.
[0103] In another aspect of the disclosure, the process described
herein is used to create liposomal delivery vehicles, such as for
example, a lipid nanoparticle (LNP) or a liposome. In some
embodiments, the process includes 1) a reservoir to provide a
solution of one or more kinds of lipids; 2) a second reservoir to
provide an additional solution of one or more kind of lipids; 3)
conduits for the first and the second reservoirs to allow for flow
of the lipid solution; 4) a junction for mixing the lipids; and 5)
a receptacle or conduit for collecting the created liposomal
delivery vehicle. In some embodiments, the first and the second
reservoirs have the same solution of one or more kinds of
lipids.
[0104] In another aspect of the disclosure, a system for
encapsulating nucleic acids is provided. Accordingly, in some
embodiments, the system includes 1) a first reservoir to provide a
desired nucleic acids in aqueous solution; 2) a second reservoir to
provide a solution of lipids and/or lipid nanoparticles (LNPs); 3)
conduits for the first and second reservoirs to allow for flow of
nucleic acids, and lipids and/or LNPs; 4) a junction for mixing the
nucleic acids and lipids and/or LNPs; and 5) a receptacle or
conduit for collecting the mixed/encapsulated nucleic acids in
LNPs.
[0105] Controlling Flow Rate in the Gravity-Based Encapsulation
Process
[0106] Various manners of controlling liquid flow rate and the
resulting mixing process to achieve reproducible encapsulation of
nucleic acids are envisioned. In some embodiments, the liquid flow
rate is controlled by adjusting the diameter of one or more of the
reservoirs, conduits, and/or junctions. In embodiments, the
diameter of the reservoirs, conduits, and/or junctions is
controlled by the use of reservoirs, conduits and/or junctions of a
specific diameter. In embodiments, the diameter of the reservoirs,
conduits and/or junctions are adjusted by providing a constriction
in one or more of the reservoirs, conduits and/or junctions.
[0107] Thus, in embodiments, the liquid flow rate and the mixing
process are controlled by providing a constriction in the first
and/or the second reservoir. In some embodiments, the liquid flow
rate and the mixing process are controlled by providing a
constriction at the reservoir-conduit connection. In some
embodiments, the liquid flow rate and the mixing process are
controlled by providing a constriction in a conduit associated with
the first and/or second reservoir. In some embodiments, the liquid
flow rate and the mixing process are controlled by providing a
constriction at the conduit-junction connection. In some
embodiments, the liquid flow rate and the mixing process are
controlled by providing a constriction in the junction. Providing
any one or more of the above constrictions allows for fine tuning
the liquid flow rate and the mixing process.
[0108] In some embodiments, the liquid flow and mixing processes
are controlled by adjustment of a diameter of one or more of the
reservoirs, conduits, and/or junctions. Accordingly, in some
embodiments the diameter of the first and/or the second reservoir
is adjusted to a desired diameter, thus providing a first diameter
associated with the first reservoir and a second diameter
associated with the second reservoir. Any method to achieve the
desired diameter is envisioned, including for example use of a
reservoir manufactured to have a certain diameter, or placement of
a constriction on a reservoir to achieve the desired diameter. In
some embodiments, the diameter of the first reservoir is about
between 1 cm-100 cm, 100 cm-1 dm, 1 dm-100 dm, 10 m-50 m. In some
embodiments, the diameter of the second reservoir is between 1
cm-100 cm, 100 cm-1 dm, 1 dm-100 dm, 10 m-50 m. In some
embodiments, the first and the second reservoir have the same
diameter. In some embodiments, the first reservoir has a larger
diameter than the second reservoir. In some embodiments, the second
reservoir has a larger diameter than the first reservoir. In some
embodiments, the diameter of the first reservoir is 1.2.times.,
1.5.times., 1.8.times., 2.0.times., 2.5.times. or greater than the
diameter of the second reservoir. In some embodiments, the diameter
of the second reservoir is 1.2.times., 1.5.times., 1.8.times.,
2.0.times., 2.5.times. or greater than the diameter of the second
reservoir.
[0109] In some embodiments, the liquid flow and mixing processes
are controlled by adjustment of a diameter of one or more of the
conduits. In some embodiments, the process uses a first conduit and
a second conduit as depicted in FIG. 1, thus providing a first
diameter associated with the first conduit and a second diameter
associated with the second conduit. The desired diameter of the
first and the second conduits is achieved by any method known in
the art, for example by use of a conduit having a specific
manufactured diameter, or by placement of a constriction on a
conduit to achieve a desired conduit diameter. In some embodiments,
the diameter of a first conduit is about between 0.1 mm-1 mm, 1
mm-100 mm, 100 mm-1 cm, 1 cm-100 cm, 100 cm-1 dm, 1 dm-100 dm, 100
dm-1 m. In some embodiments, the first conduit is about between 0.1
mm-1 mm, 1 mm-100 mm, 100 mm-1 cm, 1 cm-100 cm. In some
embodiments, the diameter of the second conduit is about between
0.1 mm-1 mm, 1 mm-100 mm, 100 mm-1 cm, 1 cm-100 cm, 100 cm-1 dm, 1
dm-100 dm, 100 dm-1 m. In some embodiments, the diameter of the
second conduit is about between 0.1 mm-1 mm, 1 mm-100 mm, 100 mm-1
cm, and 1 cm-100 cm. In some embodiments, the first and the second
conduit have the same diameter. In embodiments, the first conduit
has a larger diameter than the second conduit. In embodiments, the
second conduit has a larger diameter than the first conduit. In
embodiments, the diameter of the first conduit is 1.2.times.,
1.5.times., 1.8.times., 2.0.times., 2.5.times. or greater than the
diameter of the second conduit. In embodiments, the diameter of the
second conduit is 1.2.times., 1.5.times., 1.8.times., 2.0.times.,
2.5.times. or greater than the diameter of the second conduit.
[0110] In some embodiments, the liquid flow and mixing processes
are controlled by adjustment of a diameter at the reservoir-conduit
junction. In some embodiments, the process has a first
reservoir-conduit junction and a second reservoir-conduit junction
associated with reservoir 1 and reservoir 2, respectively and as
depicted in FIG. 1, thus providing a first diameter associated with
the first reservoir-conduit junction and a second diameter
associated with the second reservoir-conduit junction. The desired
diameter of the first and the second reservoir-conduit junction is
achieved by any method known in the art, for example by use of a
reservoir-conduit junction having a specific manufactured diameter,
or by placement of a constriction on a reservoir-conduit junction
to achieve a desired reservoir-conduit junction diameter. In some
embodiments, the diameter of a first reservoir-conduit junction is
about between 0.1 mm-1 mm, 1 mm-100 mm, 100 mm-1 cm, 1 cm-100 cm,
100 cm-1 dm, 1 dm-100 dm, 100 dm-1 m. In some embodiments, the
first reservoir-conduit is about between 0.1 mm-1 mm, 1 mm-100 mm,
100 mm-1 cm, 1 cm-100 cm. In some embodiments, the diameter of the
second reservoir-conduit is about between 0.1 mm-1 mm, 1 mm-100 mm,
100 mm-1 cm, 1 cm-100 cm, 100 cm-1 dm, 1 dm-100 dm, 100 dm-1 m. In
some embodiments, the diameter of the second reservoir-conduit is
about between 0.1 mm-1 mm, 1 mm-100 mm, 100 mm-1 cm, and 1 cm-100
cm. In some embodiments, the first and the second reservoir-conduit
have the same diameter. In some embodiments, the first
reservoir-conduit has a larger diameter than the second
reservoir-conduit. In some embodiments, the second
reservoir-conduit has a larger diameter than the first
reservoir-conduit. In some embodiments, the diameter of the first
reservoir-conduit is 1.2.times., 1.5.times., 1.8.times.,
2.0.times., 2.5.times. or greater than the diameter of the second
reservoir-conduit. In some embodiments, the diameter of the second
reservoir-conduit is 1.2.times., 1.5.times., 1.8.times.,
2.0.times., 2.5.times. or greater than the diameter of the second
reservoir-conduit.
[0111] In some embodiments, the liquid flow and mixing processes
are controlled by adjustment of a diameter at the junction. In some
embodiments, the junction is a T-shaped connector or a Y-shaped
connector. In some embodiments, the junction is a T-shaped
connector. In some embodiments, the junction is a Y-shaped
connector. In some embodiments, the process has a junction that has
a first diameter which connects to the first conduit, and the
junction has a second diameter which connects to the second
conduit. The junction and the associated connections with the first
and the second conduits are depicted in FIG. 1. The desired
diameter of the first and the second diameter of the junction is
achieved by any method known in the art, for example by use of a
junction having a specific manufactured first and/or second
diameter, or by placement of a constriction at the first and/or the
second diameter of the junction to achieve a desired
reservoir-conduit junction diameter. In some embodiments, the first
diameter of the junction is about between 0.1 mm-1 mm, 1 mm-100 mm,
100 mm-1 cm, 1 cm-100 cm, 100 cm-1 dm, 1 dm-100 dm, 100 dm-1 m. In
some embodiments, the first diameter of the junction is about
between 0.1 mm-1 mm, 1 mm-100 mm, 100 mm-1 cm, 1 cm-100 cm. In some
embodiments, the second diameter of the junction is about between
0.1 mm-1 mm, 1 mm-100 mm, 100 mm-1 cm, 1 cm-100 cm, 100 cm-1 dm, 1
dm-100 dm, 100 dm-1 m. In some embodiments, the second diameter of
the junction is about between 0.1 mm-1 mm, 1 mm-100 mm, 100 mm-1
cm, and 1 cm-100 cm. In some embodiments, the first and second
diameter of the junction are the same. In some embodiments, the
first diameter of the junction has a larger diameter than the
second diameter of the junction. In some embodiments, the second
diameter of the junction has a larger diameter than the first
diameter of the junction. In some embodiments, the first diameter
of the junction is 1.2.times., 1.5.times., 1.8.times., 2.0.times.,
2.5.times. or greater than the diameter of the second diameter of
the junction. In some embodiments, the diameter of the second
diameter of the junction is 1.2.times., 1.5.times., 1.8.times.,
2.0.times., 2.5.times. or greater than the diameter of the second
diameter of the junction.
[0112] Adjusting the first and second diameters as described above
allows for a controlled flow rate and thus a controlled mixing
process. Adjustment of the first diameter allows for a resultant
first flow rate, and an adjustment of the second diameter allows
for a resultant second flow rate. In some embodiments, the first
diameter is identical to the second diameter. In some embodiments,
the first diameter is different than the second diameter. In some
embodiments, the first diameter is larger than the second diameter.
In some embodiments, the first diameter is larger than the second
diameter in an amount that provides a first controlled flow rate to
second controlled flow rate. In some embodiments, the first
controlled flow rate to second controlled flow rate ratio is about
1:1, 2:1, 3:1, 4:1, 5:1, 10:1, 1:1 or greater, 2:1 or greater, 3:1
or greater, or 4:1 or greater, or 5:1 or greater, or 10:1 or
greater. In some embodiments, the first controlled flow rate to
second controlled flow rate ratio is about 1:1. In some
embodiments, the first controlled flow rate to second controlled
flow rate ratio is about 1:1 or greater. In some embodiments, the
first controlled flow rate to second controlled flow rate ratio is
about 2:1. In some embodiments, the first controlled flow rate to
second controlled flow rate ratio is about 2:1 or greater. In some
embodiments, the first controlled flow rate to second controlled
flow rate ratio is about 3:1. In some embodiments, the first
controlled flow rate to second controlled flow rate ratio is about
3:1 or greater. In some embodiments, the first controlled flow rate
to second controlled flow rate ratio is about 4:1. In some
embodiments, the first controlled flow rate to second controlled
flow rate ratio is about 4:1 or greater. In some embodiments, the
first controlled flow rate to second controlled flow rate ratio is
about 5:1. In some embodiments, the first controlled flow rate to
second controlled flow rate ratio is about 5:1 or greater. In some
embodiments, the first controlled flow rate to second controlled
flow rate ratio is about 10:1. In some embodiments, the first
controlled flow rate to second controlled flow rate ratio is about
10:1 or greater. In some embodiments, the first controlled flow
rate to second controlled flow rate ratio for Process A is about
1:1. In some embodiments, the first controlled flow rate to second
controlled flow rate ratio for Process A is about 2:1. In some
embodiments, the first controlled flow rate to second controlled
flow rate ratio for Process A is about 3:1. In some embodiments,
the first controlled flow rate to second controlled flow rate ratio
for Process A is about 4:1. In some embodiments, the first
controlled flow rate to second controlled flow rate ratio for
Process A is about 5:1. In some embodiments, the first controlled
flow rate to second controlled flow rate ratio for Process A is
about 1:1. In some embodiments, the first controlled flow rate to
second controlled flow rate ratio for Process B is about 1:1. In
some embodiments, the first controlled flow rate to second
controlled flow rate ratio for Process B is about 2:1. In some
embodiments, the first controlled flow rate to second controlled
flow rate ratio for Process B is about 3:1. In some embodiments,
the first controlled flow rate to second controlled flow rate ratio
for Process B is about 4:1.
[0113] In some embodiments, the second diameter is larger than the
first diameter in an amount that provides a first controlled flow
rate to second controlled flow rate. In some embodiments, the
second controlled flow rate to first controlled flow rate ratio is
about 1:1, 2:1, 3:1, 4:1, 5:1, 10:1, 1:1 or greater, 2:1 or
greater, 3:1 or greater, or 4:1 or greater, or 5:1 or greater, or
10:1 or greater. In some embodiments, the second controlled flow
rate to first controlled flow rate ratio is about 1:1. In some
embodiments, the second controlled flow rate to first controlled
flow rate ratio is about 1:1 or greater. In some embodiments, the
second controlled flow rate to first controlled flow rate ratio is
about 2:1. In some embodiments, the second controlled flow rate to
first controlled flow rate ratio is about 2:1 or greater. In some
embodiments, the second controlled flow rate to first controlled
flow rate ratio is about 3:1. In some embodiments, the second
controlled flow rate to first controlled flow rate ratio is about
3:1 or greater. In some embodiments, the second controlled flow
rate to first controlled flow rate ratio is about 4:1. In some
embodiments, the second controlled flow rate to first controlled
flow rate ratio is about 4:1 or greater. In some embodiments, the
second controlled flow rate to first controlled flow rate ratio is
about 5:1. In some embodiments, the second controlled flow rate to
first controlled flow rate ratio is about 5:1 or greater. In some
embodiments, the second controlled flow rate to first controlled
flow rate ratio is about 10:1. In some embodiments, the second
controlled flow rate to first controlled flow rate ratio is about
10:1 or greater.
[0114] Adjustment of the diameters as described herein is used to
produce a controlled flow rate to achieve a desired mixing of the
nucleic acids with lipids. In some embodiments, a first controlled
flow rate is achieved in which the flow rate is about 0.1-1 mL/min,
1-150 mL/min, 150-250 mL/min, 250-500 mL/min, 500-1000 mL/min,
1000-2000 mL/min, 2000-3000 mL/min, 3000-4000 mL/min or 4000-5000
mL/min. In some embodiments, the first controlled flow rate is
about 0.1-1 mL/min. In some embodiments, the first controlled flow
rate is about 1-150 mL/min. In some embodiments, the first
controlled flow rate is about 150-250 mL/min. In some embodiments,
the first controlled flow rate is about 250-500 mL/min. In some
embodiments, the first controlled flow rate is about 500-1000
mL/min. In some embodiments, the first controlled flow rate is
about 1000-2000 mL/min. In some embodiments, the first controlled
flow rate is about 2000-3000 mL/min. In some embodiments, the first
controlled flow rate is about 3000-4000 mL/min. In some
embodiments, the first controlled flow rate is about 4000-5000
mL/min. In some embodiments, the first controlled flow rate is
about between 150 and 250 mL/min (e.g. about 150 mL/min, 155
mL/min, 160 mL/min, 165 mL/min, 170 mL/min, 175 mL/min, 180 mL/min,
185 mL/min, 190 mL/min, 195 mL/min, 200 mL/min, 205 mL/min, 210
mL/min, 215 mL/min, 220 mL/min, 225 mL/min, 230 mL/min, 235 mL/min,
240 mL/min, 245 mL/min, or 250 mL/min).
[0115] In some embodiments, a second controlled flow rate is
achieved in which the flow rate is about 0.1-1 mL/min, 1-150
mL/min, 150-250 mL/min, 250-500 mL/min, 500-1000 mL/min, 1000-2000
mL/min, 2000-3000 mL/min, 3000-4000 mL/min or 4000-5000 mL/min. In
some embodiments, the second controlled flow rate is about 0.1-1
mL/min. In some embodiments, the second controlled flow rate is
about 1-150 mL/min. In some embodiments, the second controlled flow
rate is about 150-250 mL/min. In some embodiments, the second
controlled flow rate is about 250-500 mL/min. In some embodiments,
the second controlled flow rate is about 500-1000 mL/min. In some
embodiments, the second controlled flow rate is about 1000-2000
mL/min. In some embodiments, the second controlled flow rate is
about 2000-3000 mL/min. In some embodiments, the second controlled
flow rate is about 3000-4000 mL/min. In some embodiments, the
second controlled flow rate is about 4000-5000 mL/min. In some
embodiments, the second controlled flow rate is about between 25
and 75 mL/min (e.g. about 25 mL/min, 26 mL/min, 27 mL/min, 28
mL/min, 29 mL/min, 30 mL/min, 31 mL/min. 32 mL/min, 33 mL/min, 34
mL/min, 35 mL/min, 36 mL/min, 37 mL/min, 38 mL/min, 39 mL/min, 40
mL/min, 41 mL/min, 42 mL/min, 43 mL/min, 44 mL/min, 45 mL/min, 46
mL/min, 47 mL/min, 48 mL/min, 49 mL/min, 50 mL/min, 51 mL/min, 52
mL/min, 53 mL/min, 54 mL/min, 55 mL/min, 56 mL/min, 57 mL/min, 58
mL/min, 59 mL/min, 60 mL/min, 61 mL/min, 62 mL/min, 63 mL/min, 64
mL/min, 65 mL/min, 66 mL/min, 67 mL/min, 68 mL/min, 69 mL/min, 70
mL/min, 71 mL/min, 72 mL/min, 73 mL/min, 74 mL/min, or 75 mL/min)
In some embodiments, the first controlled flow rate is about 50
mL/min.
[0116] In some embodiments, the first controlled flow rate to
second controlled flow rate is at a ratio of about 1:1, 2:1, 3:1,
4:1, 5:1, 10:1, 1:1 or greater, 2:1 or greater, 3:1 or greater, or
4:1 or greater, or 5:1 or greater, or 10:1 or greater. Accordingly,
in some embodiments, the first controlled flow rate to second
controlled flow rate is at a ratio of about 1:1. In some
embodiments, the first controlled flow rate to second controlled
flow rate is at a ratio of about 2:1. In some embodiments, the
first controlled flow rate to second controlled flow rate is at a
ratio of about 3:1. In some embodiments, the first controlled flow
rate to second controlled flow rate is at a ratio of about 4:1. In
some embodiments, the first controlled flow rate to second
controlled flow rate is at a ratio of about 5:1. In some
embodiments, the first controlled flow rate to second controlled
flow rate is at a ratio of about 10:1. In some embodiments, the
first controlled flow rate to second controlled flow rate is at a
ratio of about 1:1 or greater. In some embodiments, the first
controlled flow rate to second controlled flow rate is at a ratio
of about 2:1 or greater. In some embodiments, the first controlled
flow rate to second controlled flow rate is at a ratio of about 3:1
or greater. In some embodiments, the first controlled flow rate to
second controlled flow rate is at a ratio of about 4:1 or greater.
In some embodiments, the first controlled flow rate to second
controlled flow rate is at a ratio of about 5:1 or greater. In some
embodiments, the first controlled flow rate to second controlled
flow rate is at a ratio of about 10:1 or greater.
[0117] In some embodiments, the second controlled flow rate to
first controlled flow rate is at a ratio of about 1:1, 2:1, 3:1,
4:1, 5:1, 10:1, 1:1 or greater, 2:1 or greater, 3:1 or greater, or
4:1 or greater, or 5:1 or greater, or 10:1 or greater. Accordingly,
in some embodiments, the second controlled flow rate to first
controlled flow rate is at a ratio of about 1:1. In some
embodiments, the second controlled flow rate to first controlled
flow rate is at a ratio of about 2:1. In some embodiments, the
second controlled flow rate to first controlled flow rate is at a
ratio of about 3:1. In some embodiments, the second controlled flow
rate to first controlled flow rate is at a ratio of about 4:1. In
some embodiments, t the second controlled flow rate to first
controlled flow rate is at a ratio of about 5:1. In some
embodiments, the second controlled flow rate to first controlled
flow rate is at a ratio of about 10:1. In some embodiments, the
second controlled flow rate to first controlled flow rate is at a
ratio of about 1:1 or greater. In some embodiments, the second
controlled flow rate to first controlled flow rate is at a ratio of
about 2:1 or greater. In some embodiments, the second controlled
flow rate to first controlled flow rate is at a ratio of about 3:1
or greater. In some embodiments, the second controlled flow rate to
first controlled flow rate is at a ratio of about 4:1 or greater.
In some embodiments, the second controlled flow rate to first
controlled flow rate is at a ratio of about 5:1 or greater. In some
embodiments, the second controlled flow rate to first controlled
flow rate is at a ratio of about 10:1 or greater.
[0118] In some embodiments, a first flow rate is controlled and a
second flow rate is not controlled. For example, in some
embodiments, one flow stream is mixed with controlled flow rate
into a reservoir containing a desired component. Thus, in some
embodiments a flow rate stream of a nucleic acid solution is
controlled. In some embodiments, a flow rate stream of a lipid
solution or LNP is controlled. In some embodiments, the flow rate
stream of a nucleic acid solution is controlled and the flow rate
stream of a lipid solution or LNP is controlled. In some
embodiments, the first flow comprises a nucleotide. In some
embodiments, the second flow comprises a lipid solution or LNP. In
some embodiments, the first flow and the second flow comprise a
nucleic acid. In some embodiments, the nucleic acid can be DNA or
RNA. In some embodiments, the first flow and the second flow
comprise a lipid solution or a LNP.
[0119] Gravity-Based Encapsulation Process--Controlled Mixing
[0120] In some embodiments, a lipid solution containing dissolved
lipids, and an aqueous or buffer solution are mixed into a solution
such that the lipids can form nanoparticles without mRNA (or empty
preformed lipid nanoparticles). In some embodiments, an mRNA
solution and a preformed lipid nanoparticle solution are mixed into
a solution such that the mRNA becomes encapsulated in the lipid
nanoparticle. Such a solution is also referred to as a formulation
or encapsulation solution. A suitable formulation or encapsulation
solution includes a solvent such as ethanol. For example, a
suitable formulation or encapsulation solution includes about 10%
ethanol, about 15% ethanol, about 20% ethanol, about 25% ethanol,
about 30% ethanol, about 35% ethanol, or about 40% ethanol.
[0121] Controlling the flow rate in the process described herein
allows for reproducible encapsulation of nucleic acids in lipids.
The controlled flow rate in the process described herein also
allows for reproducible production of lipid nanoparticles (LNPs).
The controlled flow rate achieves reproducible mixing of the
components of a first flow stream and a second flow stream. In some
embodiments, the mixing of the first flow stream and the second
flow stream occurs at a junction as depicted in FIG. 1. In some
embodiments, the junction is a T-connector (also known as "Tee"
connector) or a "Y" connector. In some embodiments, the junction is
a T-connector. In some embodiments, the junction is a Y connector.
In some embodiments, the T-connector has symmetrical arms,
asymmetrical arms or arms comprising different diameters. In some
embodiments, the T-connector has symmetrical arms. In some
embodiments, the T-connector has asymmetrical arms. In some
embodiments, the T-connector has arms comprising different
diameters. In some embodiments, the Y-connector has symmetrical
arms, asymmetrical arms or arms comprising different diameters. In
some embodiments, the Y-connector has symmetrical arms. In some
embodiments, the Y-connector has asymmetrical arms. In some
embodiments, the Y-connector has arms comprising different
diameters. In some embodiments, the flow rate and resultant mixing
process is controlled by the diameter of one or more arms of the
connector at the junction. In some embodiments, the diameter of an
arm of the connector is controlled by a constriction placed over
the arm. In some embodiments, the diameter of the connector arm is
premade. In some embodiments, flow rate is controlled by a stopcock
that is placed at one or more arms of the connector.
[0122] In some embodiments, a first conduit comprising a nucleic
acid is connected at one arm of a T- or a Y-connector, and a second
conduit comprising a lipid solution or LNP are connected at another
arm of the T-connector or a Y-connector junction, thereby mixing
the solutions from the first conduit and the second conduit at the
T-connector or Y-connector junction. The mixing of the solutions
results in the encapsulation of the nucleic acid in lipid
nanoparticles.
[0123] In some embodiments, a first conduit comprising an mRNA is
connected at one arm of a T- or a Y-connector, and a second conduit
comprising a lipid solution or LNP are connected at another arm of
the T-connector or a Y-connector junction, thereby mixing the
solutions from the first conduit and the second conduit at the
T-connector or Y-connector junction. The mixing of the solutions
results in the encapsulation of the mRNA in lipid
nanoparticles.
[0124] In some embodiments, a first conduit comprising a first
lipid solution is connected at one arm of a T- or a Y-connector,
and a second conduit comprising a second lipid solution is
connected at another arm of the T-connector or a Y-connector
junction, thereby mixing the solutions from the first conduit and
the second conduit at the T-connector or Y-connector junction. The
mixing of the solutions results in the production of liquid
nanoparticles (LNPs). In some embodiments, the first lipid solution
and the second lipid solution are the same. In some embodiments,
the first lipid solution is a cationic lipid solution and the
second lipid solution is a non-cationic (also referred to herein as
"helper lipid") lipid solution. In some embodiments, the first
lipid solution is a cationic lipid solution and the second lipid
solution is a PEGylated lipid solution. In some embodiments, the
first lipid solution is a non-cationic lipid solution and the
second lipid solution is a PEGylated lipid solution. In some
embodiments, the first lipid solution is a cationic lipid solution
and the second lipid solution is a cholesterol-based lipid
solution. In some embodiments, the first lipid solution is a
cholesterol-based lipid solution and the second lipid solution is a
non-cationic lipid solution. In some embodiments, the first lipid
solution is a cholesterol-based lipid solution and the second lipid
solution is a PEGylated lipid solution. In some embodiments, the
lipid solution comprises pre-formed lipid nanoparticles. In some
embodiments, the lipid solution comprises a suspension of
pre-formed lipid nanoparticles.
[0125] In some embodiments, the first flow stream comprises an mRNA
solution and the second flow stream comprises a lipid solution. In
embodiments, the first flow stream comprises about between 50-99%
water (e.g. 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or
99% water). In some embodiments, the first flow stream comprises
about 50% water. In some embodiments, the first flow stream
comprises about 55% water. In some embodiments, the first flow
stream comprises about 60% water. In some embodiments, the first
flow stream comprises about 65% water. In some embodiments, the
first flow stream comprises about 70% water. In some embodiments,
the first flow stream comprises about 75% water. In some
embodiments, the first flow stream comprises about 80% water. In
some embodiments, the first flow stream comprises about 85% water.
In some embodiments, the first flow stream comprises about 90%
water. In some embodiments, the first flow stream comprises about
95% water. In some embodiments, the first flow stream comprises
about 96% water. In some embodiments, the first flow stream
comprises about 97% water. In some embodiments, the first flow
stream comprises about 98% water. In some embodiments, the first
flow stream comprises about 99% water.
[0126] In some embodiments, the second flow stream comprises about
between 50-99% ethanol (e.g. 50, 55, 60, 65, 70, 75, 80, 85, 90,
95, 96, 97, 98, or 99% ethanol). In some embodiments, the second
flow stream comprises about 50% ethanol. In some embodiments, the
second flow stream comprises about 55% ethanol. In some
embodiments, the second flow stream comprises about 60% ethanol. In
some embodiments, the second flow stream comprises about 65%
ethanol. In some embodiments, the second flow stream comprises
about 70% ethanol. In some embodiments, the second flow stream
comprises about 75% ethanol. In some embodiments, the second flow
stream comprises about 80% ethanol. In some embodiments, the second
flow stream comprises about 85% ethanol. In some embodiments, the
second flow stream comprises about 90% ethanol. In some
embodiments, the second flow stream comprises about 95% ethanol. In
some embodiments, the second flow stream comprises about 96%
ethanol. In some embodiments, the second flow stream comprises
about 97% ethanol. In some embodiments, the second flow stream
comprises about 98% ethanol. In some embodiments, the second flow
stream comprises about 99% ethanol.
[0127] In some embodiments, the mixing of the first stream and the
second stream results in a mixture comprising about 5-60% ethanol
(e.g. about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 60% ethanol).
In embodiments, the mixing of the first stream and the second
stream results in a mixture comprising about 5% ethanol. In
embodiments, the mixing of the first stream and the second stream
results in a mixture comprising about 10% ethanol. In embodiments,
the mixing of the first stream and the second stream results in a
mixture comprising about 15% ethanol. In embodiments, the mixing of
the first stream and the second stream results in a mixture
comprising about 20% ethanol. In embodiments, the mixing of the
first stream and the second stream results in a mixture comprising
about 25% ethanol. In embodiments, the mixing of the first stream
and the second stream results in a mixture comprising about 30%
ethanol. In embodiments, the mixing of the first stream and the
second stream results in a mixture comprising about 35% ethanol. In
embodiments, the mixing of the first stream and the second stream
results in a mixture comprising about 40% ethanol. In embodiments,
the mixing of the first stream and the second stream results in a
mixture comprising about 45% ethanol. In embodiments, the mixing of
the first stream and the second stream results in a mixture
comprising about 50% ethanol. In embodiments, the mixing of the
first stream and the second stream results in a mixture comprising
about 55% ethanol. In embodiments, the mixing of the first stream
and the second stream results in a mixture comprising about 60%
ethanol.
[0128] The process described herein results in reproducible, high
nucleic acid encapsulation efficiencies. In some embodiments, using
the process described herein mRNA is encapsulated in lipids at an
efficiency of about between 60-100% (e.g. about 75, 76, 77, 78, 79,
80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 98,
or 99% encapsulation efficiency). In some embodiments, mRNA is
encapsulated in lipids at an efficiency of about 60%. In some
embodiments, mRNA is encapsulated in lipids at an efficiency of
about 65%. In some embodiments, mRNA is encapsulated in lipids at
an efficiency of about 70%. In some embodiments, mRNA is
encapsulated in lipids at an efficiency of about 75%. In some
embodiments, mRNA is encapsulated in lipids at an efficiency of
about 76%. In some embodiments, mRNA is encapsulated in lipids at
an efficiency of about 77%. In some embodiments, mRNA is
encapsulated in lipids at an efficiency of about 78%. In some
embodiments, mRNA is encapsulated in lipids at an efficiency of
about 79%. In some embodiments, mRNA is encapsulated in lipids at
an efficiency of about 80%. In some embodiments, mRNA is
encapsulated in lipids at an efficiency of about 81%. In some
embodiments, mRNA is encapsulated in lipids at an efficiency of
about 82%. In some embodiments, mRNA is encapsulated in lipids at
an efficiency of about 83%. In some embodiments, mRNA is
encapsulated in lipids at an efficiency of about 84%. In some
embodiments, mRNA is encapsulated in lipids at an efficiency of
about 85%. In some embodiments, mRNA is encapsulated in lipids at
an efficiency of about 86%. In some embodiments, mRNA is
encapsulated in lipids at an efficiency of about 87%. In some
embodiments, mRNA is encapsulated in lipids at an efficiency of
about 88%. In some embodiments, mRNA is encapsulated in lipids at
an efficiency of about 89%. In some embodiments, mRNA is
encapsulated in lipids at an efficiency of about 90%. In some
embodiments, mRNA is encapsulated in lipids at an efficiency of
about 91%. In some embodiments, mRNA is encapsulated in lipids at
an efficiency of about 92%. In some embodiments, mRNA is
encapsulated in lipids at an efficiency of about 93%. In some
embodiments, mRNA is encapsulated in lipids at an efficiency of
about 94%. In some embodiments, mRNA is encapsulated in lipids at
an efficiency of about 95%.
[0129] The encapsulated nucleic acids using the process described
herein have a nanoparticle size of about 40-150 nm. In some
embodiments, the encapsulation efficiency is about 60%. In some
embodiments, the encapsulation efficiency is about 65%. In some
embodiments, the encapsulation efficiency is about 70%. In some
embodiments, the encapsulation efficiency is about 75%. In some
embodiments, the encapsulation efficiency is about 80%. In some
embodiments, the encapsulation efficiency is about 85%. In some
embodiments, the encapsulation efficiency is about 90%. In some
embodiments, the encapsulation efficiency is about 95%. In some
embodiments, the encapsulation efficiency is about 96%. In some
embodiments, the encapsulation efficiency is about 97%. In some
embodiments, the encapsulation efficiency is about 98%. In some
embodiments, the encapsulation efficiency is about 99%.
[0130] The process described herein has high amounts of mRNA
recovery. In embodiments, the process results in at least about 50,
55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% recovery of
mRNA. In embodiments, the process results in at least about 50%
recover of mRNA. In embodiments, the process results in at least
about 55% recover of mRNA. In embodiments, the process results in
at least about 60% recover of mRNA. In embodiments, the process
results in at least about 65% recover of mRNA. In embodiments, the
process results in at least about 70% recover of mRNA. In
embodiments, the process results in at least about 75% recover of
mRNA. In embodiments, the process results in at least about 80%
recover of mRNA. In embodiments, the process results in at least
about 85% recover of mRNA. In embodiments, the process results in
at least about 90% recover of mRNA. In embodiments, the process
results in at least about 95% recover of mRNA. In embodiments, the
process results in at least about 96% recover of mRNA. In
embodiments, the process results in at least about 97% recover of
mRNA. In embodiments, the process results in at least about 98%
recover of mRNA. In embodiments, the process results in at least
about 99% recover of mRNA.
[0131] In some embodiments, the process results in at least about
0.1 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 100 mg, 500 mg, or 1,000 mg of
encapsulated mRNA. In some embodiments, the process results in at
least about 0.1 mg of encapsulated mRNA. In some embodiments, the
process results in at least about 0.5 mg of encapsulated mRNA. In
some embodiments, the process results in at least about 1 mg of
encapsulated mRNA. In some embodiments, the process results in at
least about 5 mg of encapsulated mRNA. In some embodiments, the
process results in at least about 10 mg of encapsulated mRNA. In
some embodiments, the process results in at least about 100 mg of
encapsulated mRNA. In some embodiments, the process results in at
least about 500 mg of encapsulated mRNA. In some embodiments, the
process results in at least about 1000 mg of encapsulated mRNA.
[0132] In some embodiments, the mixing of the first and the second
streams occurs simultaneously. In some embodiments, the mixing of
the first and second streams occurs asynchronously.
[0133] In some embodiments, the use of the process disclosed herein
results in lipid nanoparticles that do not require further
purification.
[0134] High Throughput Formulation
[0135] The gravity-based encapsulation process is configurable such
that multiple flow streams are envisioned. Multiple flow streams
allows for a high throughput process. In embodiments, an assembly
line approach is achieved wherein liquids are added to pairs of
reservoirs at the same time, followed by the addition of liquids to
the next pairs of reservoirs in succession. An embodiment of the
gravity-based encapsulation process which depicts multiple streams
is shown in FIG. 7 and FIG. 8. In some embodiments, multiple pairs
of first conduit streams (Flow 1) and second conduit streams (Flow
2) are used. For example, in some embodiments the process comprises
at least about 1 pair, 5 pairs, 10 pairs, 20 pairs, 30 pairs, 40
pairs, 50 pairs, 100 pairs, 150 pairs, 200 pairs, 250 pairs, or 300
pairs of first conduit streams and second conduit streams. In some
embodiments, the process comprises 1 pair of first conduit streams
and second conduit streams. In some embodiments, the process
comprises about 5 pairs of first conduit streams and second conduit
streams. In some embodiments, the process comprises about 10 pairs
of first conduit streams and second conduit streams. In some
embodiments, the process comprises about 20 pairs of first conduit
streams and second conduit streams. In some embodiments, the
process comprises about 30 pairs of first conduit streams and
second conduit streams. In some embodiments, the process comprises
about 40 pairs of first conduit streams and second conduit streams.
In some embodiments, the process comprises about 50 pairs of first
conduit streams and second conduit streams. In some embodiments,
the process comprises about 100 pairs of first conduit streams and
second conduit streams. In some embodiments, the process comprises
about 150 pairs of first conduit streams and second conduit
streams. In some embodiments, the process comprises about 200 pairs
of first conduit streams and second conduit streams. In some
embodiments, the process comprises about 250 pairs of first conduit
streams and second conduit streams. In some embodiments, the
process comprises about 300 pairs of first conduit streams and
second conduit streams.
[0136] In some embodiments, each individual first stream provides a
different nucleic acid solution. In some embodiments, each
individual first stream provides the same nucleic acid. In some
embodiments, each individual first stream provides a different mRNA
solution. In some embodiments, each individual first stream
provides the same mRNA solution. In some embodiments, each
individual second stream provides a different lipid solution. In
some embodiments, each individual second stream provide the same
lipid solution.
Messenger (mRNA)
[0137] The gravity-based encapsulation process described herein can
be used with any kind of nucleic acid. In some embodiments, the
nucleic acid is an RNA, including for example, messenger RNA
(mRNA), antisense RNA (aRNA), small interfering RNA (siRNA), CRISPR
RNA (crRNA), long noncoding RNA (lncRNA), microRNA (miRNA),
Piwi-interacting RNA (piRNA), short hairpin RNA (shRNA),
trnas-acting siRNA (tasiRNA), repeat associated siRNA (rasiRNA),
7SK RNA (7SK), enhancer RNA (eRNA), ribosomal RNA (rRNA), signal
recognition particle RNA (SRP RNA), transfer RNA (tRNA), small
nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA (SmY),
small Cajal body-specific RNA (scaRNA), and guide RNA (gRNA). In
embodiments, the RNA is mRNA.
[0138] The present invention may be used to encapsulate any mRNA.
mRNA is typically thought of as the type of RNA that carries
information from DNA to the ribosome. The existence of mRNA is
typically very brief and includes processing and translation,
followed by degradation. Typically, in eukaryotic organisms, mRNA
processing comprises the addition of a "cap" on the N-terminal (5')
end, and a "tail" on the C-terminal (3') end. A typical cap is a
7-methylguanosine cap, which is a guanosine that is linked through
a 5'-5'-triphosphate bond to the first transcribed nucleotide. The
presence of the cap is important in providing resistance to
nucleases found in most eukaryotic cells. The tail is typically a
polyadenylation event whereby a polyadenylyl moiety is added to the
3' end of the mRNA molecule. The presence of this "tail" serves to
protect the mRNA from exonuclease degradation. Messenger RNA is
translated by the ribosomes into a series of amino acids that make
up a protein.
[0139] mRNAs may be synthesized according to any of a variety of
known methods. For example, mRNAs according to the present
invention may be synthesized via in vitro transcription (IVT).
Briefly, IVT is typically performed with a linear or circular DNA
template containing a promoter, a pool of ribonucleotide
triphosphates, a buffer system that may include DTT and magnesium
ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA
polymerase), DNAse I, pyrophosphatase, and/or RNAse inhibitor. The
exact conditions will vary according to the specific
application.
[0140] In some embodiments, in vitro synthesized mRNA may be
purified before formulation and encapsulation to remove undesirable
impurities including various enzymes and other reagents used during
mRNA synthesis.
[0141] The present invention may be used to formulate and
encapsulate mRNAs of a variety of lengths. In some embodiments, the
present invention may be used to formulate and encapsulate in vitro
synthesized mRNA of or greater than about 1 kb, 1.5 kb, 2 kb, 2.5
kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb 6 kb, 7 kb, 8 kb, 9 kb, 10 kb,
11 kb, 12 kb, 13 kb, 14 kb, 15 kb, or 20 kb in length. In some
embodiments, the present invention may be used to formulate and
encapsulate in vitro synthesized mRNA ranging from about 1-20 kb,
about 1-15 kb, about 1-10 kb, about 5-20 kb, about 5-15 kb, about
5-12 kb, about 5-10 kb, about 8-20 kb, or about 8-15 kb in
length.
[0142] The present invention may be used to formulate and
encapsulate mRNA that is unmodified or mRNA containing one or more
modifications that typically enhance stability. In some
embodiments, modifications are selected from modified nucleotide,
modified sugar phosphate backbones, 5' and/or 3' untranslated
region.
[0143] In some embodiments, modifications of mRNA may include
modifications of the nucleotides of the RNA. A modified mRNA
according to the invention can include, for example, backbone
modifications, sugar modifications or base modifications. In some
embodiments, mRNAs may be synthesized from naturally occurring
nucleotides and/or nucleotide analogues (modified nucleotides)
including, but not limited to, purines (adenine (A), guanine (G))
or pyrimidines (thymine (T), cytosine (C), uracil (U)), and as
modified nucleotides analogues or derivatives of purines and
pyrimidines, such as e.g. 1-methyl-adenine, 2-methyl-adenine,
2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine,
N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine,
4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine,
1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine,
7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil
(5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil,
5-carboxymethylaminomethyl-2-thio-uracil,
5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil,
5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil,
5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester,
5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil,
5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil,
uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v),
1-methyl-pseudouracil, queosine, .beta.-D-mannosyl-queosine,
wybutoxosine, and phosphoramidates, phosphorothioates, peptide
nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine
and inosine. The preparation of such analogues is known to a person
skilled in the art e.g. from the U.S. Pat. Nos. 4,373,071,
4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679,
5,047,524, 5,132,418, 5,153,319, 5,262,530 and 5,700,642, the
disclosure of which is included here in its full scope by
reference.
[0144] Typically, mRNA synthesis includes the addition of a "cap"
on the N-terminal (5') end, and a "tail" on the C-terminal (3')
end. The presence of the cap is important in providing resistance
to nucleases found in most eukaryotic cells. The presence of a
"tail" serves to protect the mRNA from exonuclease degradation.
[0145] Thus, in some embodiments, mRNAs include a 5' cap structure.
A 5' cap is typically added as follows: first, an RNA terminal
phosphatase removes one of the terminal phosphate groups from the
5' nucleotide, leaving two terminal phosphates; guanosine
triphosphate (GTP) is then added to the terminal phosphates via a
guanylyl transferase, producing a 5'5'5 triphosphate linkage; and
the 7-nitrogen of guanine is then methylated by a
methyltransferase. 2'-O-methylation may also occur at the first
base and/or second base following the 7-methyl guanosine
triphosphate residues. Examples of cap structures include, but are
not limited to, m7GpppNp-RNA, m7GpppNmp-RNA and m7GpppNmpNmp-RNA
(where m indicates 2'-Omethyl residues).
[0146] In some embodiments, mRNAs include a 5' and/or 3'
untranslated region. In some embodiments, a 5' untranslated region
includes one or more elements that affect an mRNA's stability or
translation, for example, an iron responsive element. In some
embodiments, a 5' untranslated region may be between about 50 and
500 nucleotides in length.
[0147] In some embodiments, a 3' untranslated region includes one
or more of a polyadenylation signal, a binding site for proteins
that affect a mRNA's stability of location in a cell, or one or
more binding sites for miRNAs. In some embodiments, a 3'
untranslated region may be between 50 and 500 nucleotides in length
or longer.
[0148] While mRNA provided from in vitro transcription reactions
may be desirable in some embodiments, other sources of mRNA are
contemplated as within the scope of the invention including mRNA
produced from bacteria, fungi, plants, and/or animals.
[0149] In some embodiments, the mRNA used in the process described
herein is unmodified. In some embodiments, the mRNA used in the
process disclosed herein is modified. In some embodiments, the mRNA
used in the process described herein is codon-optimized.
[0150] The present invention may be used to formulate and
encapsulate mRNAs encoding a variety of proteins.
mRNA Solution
[0151] mRNA may be provided in a solution to be mixed with a lipid
solution such that the mRNA may be encapsulated in lipid
nanoparticles. A suitable mRNA solution may be any aqueous solution
containing mRNA to be encapsulated at various concentrations. For
example, a suitable mRNA solution may contain a mRNA at a
concentration of or greater than about 0.01 mg/ml, 0.05 mg/ml, 0.06
mg/ml, 0.07 mg/ml, 0.08 mg/ml, 0.09 mg/ml, 0.1 mg/ml, 0.15 mg/ml,
0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml,
0.8 mg/ml, 0.9 mg/ml, or 1.0 mg/ml. In some embodiments, a suitable
mRNA solution may contain a mRNA at a concentration ranging from
about 0.01-1.0 mg/ml, 0.01-0.9 mg/ml, 0.01-0.8 mg/ml, 0.01-0.7
mg/ml, 0.01-0.6 mg/ml, 0.01-0.5 mg/ml, 0.01-0.4 mg/ml, 0.01-0.3
mg/ml, 0.01-0.2 mg/ml, 0.01-0.1 mg/ml, 0.05-1.0 mg/ml, 0.05-0.9
mg/ml, 0.05-0.8 mg/ml, 0.05-0.7 mg/ml, 0.05-0.6 mg/ml, 0.05-0.5
mg/ml, 0.05-0.4 mg/ml, 0.05-0.3 mg/ml, 0.05-0.2 mg/ml, 0.05-0.1
mg/ml, 0.1-1.0 mg/ml, 0.2-0.9 mg/ml, 0.3-0.8 mg/ml, 0.4-0.7 mg/ml,
or 0.5-0.6 mg/ml. In some embodiments, a suitable mRNA solution may
contain an mRNA at a concentration up to about 5.0 mg/ml, 4.0
mg/ml, 3.0 mg/ml, 2.0 mg/ml, 1.0 mg/ml, 0.09 mg/ml, 0.08 mg/ml,
0.07 mg/ml, 0.06 mg/ml, or 0.05 mg/ml.
[0152] Typically, a suitable mRNA solution may also contain a
buffering agent and/or salt. Generally, buffering agents can
include HEPES, ammonium sulfate, sodium bicarbonate, sodium
citrate, sodium acetate, potassium phosphate and sodium phosphate.
In some embodiments, suitable concentration of the buffering agent
may range from about 0.1 mM to 100 mM, 0.5 mM to 90 mM, 1.0 mM to
80 mM, 2 mM to 70 mM, 3 mM to 60 mM, 4 mM to 50 mM, 5 mM to 40 mM,
6 mM to 30 mM, 7 mM to 20 mM, 8 mM to 15 mM, or 9 to 12 mM. In some
embodiments, suitable concentration of the buffering agent is or
greater than about 0.1 mM, 0.5 mM, 1 mM, 2 mM, 4 mM, 6 mM, 8 mM, 10
mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM.
[0153] Exemplary salts can include sodium chloride, magnesium
chloride, and potassium chloride. In some embodiments, suitable
concentration of salts in a mRNA solution may range from about 1 mM
to 500 mM, 5 mM to 400 mM, 10 mM to 350 mM, 15 mM to 300 mM, 20 mM
to 250 mM, 30 mM to 200 mM, 40 mM to 190 mM, 50 mM to 180 mM, 50 mM
to 170 mM, 50 mM to 160 mM, 50 mM to 150 mM, or 50 mM to 100 mM.
Salt concentration in a suitable mRNA solution is or greater than
about 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM,
80 mM, 90 mM, or 100 mM.
[0154] In some embodiments, a suitable mRNA solution may have a pH
ranging from about 3.5-6.5, 3.5-6.0, 3.5-5.5., 3.5-5.0, 3.5-4.5,
4.0-5.5, 4.0-5.0, 4.0-4.9, 4.0-4.8, 4.0-4.7, 4.0-4.6, or 4.0-4.5.
In some embodiments, a suitable mRNA solution may have a pH of or
no greater than about 3.5, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,
4.8, 4.9, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.1, 6.3, and 6.5.
[0155] Various methods may be used to prepare an mRNA solution
suitable for the present invention. In some embodiments, mRNA may
be directly dissolved in a buffering solution described herein. In
some embodiments, an mRNA solution may be generated by mixing an
mRNA stock solution with a buffering solution prior to mixing with
a lipid solution for encapsulation. In some embodiments, an mRNA
solution may be generated by mixing an mRNA stock solution with a
buffering solution immediately before mixing with a lipid solution
for encapsulation. In some embodiments, a suitable mRNA stock
solution may contain mRNA in water at a concentration at or greater
than about 0.2 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.8 mg/ml,
1.0 mg/ml, 1.2 mg/ml, 1.4 mg/ml, 1.5 mg/ml, or 1.6 mg/ml, 2.0
mg/ml, 2.5 mg/ml, 3.0 mg/ml, 3.5 mg/ml, 4.0 mg/ml, 4.5 mg/ml, or
5.0 mg/ml.
[0156] In some embodiments, an mRNA stock solution is mixed with a
buffering solution using a pump. Exemplary pumps include but are
not limited to gear pumps, peristaltic pumps and centrifugal
pumps.
[0157] Typically, the buffering solution is mixed at a rate greater
than that of the mRNA stock solution. For example, the buffering
solution may be mixed at a rate at least 1.times., 2.times.,
3.times., 4.times., 5.times., 6.times., 7.times., 8.times.,
9.times., 10.times., 15.times., or 20.times. greater than the rate
of the mRNA stock solution. In some embodiments, a buffering
solution is mixed at a flow rate ranging between about 100-6000
ml/minute (e.g., about 100-300 ml/minute, 300-600 ml/minute,
600-1200 ml/minute, 1200-2400 ml/minute, 2400-3600 ml/minute,
3600-4800 ml/minute, 4800-6000 ml/minute, or 60-420 ml/minute). In
some embodiments, a buffering solution is mixed at a flow rate of
or greater than about 60 ml/minute, 100 ml/minute, 140 ml/minute,
180 ml/minute, 220 ml/minute, 260 ml/minute, 300 ml/minute, 340
ml/minute, 380 ml/minute, 420 ml/minute, 480 ml/minute, 540
ml/minute, 600 ml/minute, 1200 ml/minute, 2400 ml/minute, 3600
ml/minute, 4800 ml/minute, or 6000 ml/minute.
[0158] In some embodiments, a mRNA stock solution is mixed at a
flow rate ranging between about 10-600 ml/minute (e.g., about 5-50
ml/minute, about 10-30 ml/minute, about 30-60 ml/minute, about
60-120 ml/minute, about 120-240 ml/minute, about 240-360 ml/minute,
about 360-480 ml/minute, or about 480-600 ml/minute). In some
embodiments, a mRNA stock solution is mixed at a flow rate of or
greater than about 5 ml/minute, 10 ml/minute, 15 ml/minute, 20
ml/minute, 25 ml/minute, 30 ml/minute, 35 ml/minute, 40 ml/minute,
45 ml/minute, 50 ml/minute, 60 ml/minute, 80 ml/minute, 100
ml/minute, 200 ml/minute, 300 ml/minute, 400 ml/minute, 500
ml/minute, or 600 ml/minute.
Lipid Solution
[0159] According to the present invention, a lipid solution
contains a mixture of lipids suitable to form lipid nanoparticles
for encapsulation of mRNA. In some embodiments, a suitable lipid
solution is ethanol based. For example, a suitable lipid solution
may contain a mixture of desired lipids dissolved in pure ethanol
(i.e., 100% ethanol). In another embodiment, a suitable lipid
solution is isopropyl alcohol based. In another embodiment, a
suitable lipid solution is dimethylsulfoxide-based. In another
embodiment, a suitable lipid solution is a mixture of suitable
solvents including, but not limited to, ethanol, isopropyl alcohol
and dimethylsulfoxide.
[0160] A suitable lipid solution may contain a mixture of desired
lipids at various concentrations. For example, a suitable lipid
solution may contain a mixture of desired lipids at a total
concentration of or greater than about 0.1 mg/ml, 0.5 mg/ml, 1.0
mg/ml, 2.0 mg/ml, 3.0 mg/ml, 4.0 mg/ml, 5.0 mg/ml, 6.0 mg/ml, 7.0
mg/ml, 8.0 mg/ml, 9.0 mg/ml, 10 mg/ml, 15 mg/ml, 20 mg/ml, 30
mg/ml, 40 mg/ml, 50 mg/ml, or 100 mg/ml. In some embodiments, a
suitable lipid solution may contain a mixture of desired lipids at
a total concentration ranging from about 0.1-100 mg/ml, 0.5-90
mg/ml, 1.0-80 mg/ml, 1.0-70 mg/ml, 1.0-60 mg/ml, 1.0-50 mg/ml,
1.0-40 mg/ml, 1.0-30 mg/ml, 1.0-20 mg/ml, 1.0-15 mg/ml, 1.0-10
mg/ml, 1.0-9 mg/ml, 1.0-8 mg/ml, 1.0-7 mg/ml, 1.0-6 mg/ml, or 1.0-5
mg/ml. In some embodiments, a suitable lipid solution may contain a
mixture of desired lipids at a total concentration up to about 100
mg/ml, 90 mg/ml, 80 mg/ml, 70 mg/ml, 60 mg/ml, 50 mg/ml, 40 mg/ml,
30 mg/ml, 20 mg/ml, or 10 mg/ml.
[0161] Any desired lipids may be mixed at any ratios suitable for
encapsulating mRNAs. In some embodiments, a suitable lipid solution
contain a mixture of desired lipids including cationic lipids,
helper lipids (e.g. non cationic lipids and/or cholesterol lipids)
and/or PEGylated lipids. In some embodiments, a suitable lipid
solution contain a mixture of desired lipids including one or more
cationic lipids, one or more helper lipids (e.g. non cationic
lipids and/or cholesterol lipids) and one or more PEGylated
lipids.
[0162] In some embodiments, the lipid solution used in the process
includes one or more cationic lipids, one or more helper lipids,
and one or more PEG-modified lipids. In some embodiments, the lipid
solution includes one or more cationic lipids. In some embodiments,
the lipid solution includes one or more helper lipids. In some
embodiments, the lipid solution includes one or more PEG-modified
lipids. In some embodiments, the lipid solution includes one or
more cationic lipids and one or more helper lipids. In some
embodiments, the lipid solution includes one or more cationic
lipids and one or more PEG-modified lipids. In some embodiments,
the lipid solution includes one or more helper lipids and one or
more PEG-modified lipids.
[0163] Cationic Lipids
[0164] As used herein, the phrase "cationic lipids" refers to any
of a number of lipid species that have a net positive charge at a
selected pH, such as physiological pH.
[0165] Suitable cationic lipids for use in the compositions and
methods of the invention include the cationic lipids as described
in International Patent Publication WO 2010/144740, which is
incorporated herein by reference. In certain embodiments, the
compositions and methods of the present invention include a
cationic lipid,
(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl
4-(dimethylamino) butanoate, having a compound structure of:
##STR00001##
and pharmaceutically acceptable salts thereof.
[0166] Other suitable cationic lipids for use in the compositions
and methods of the present invention include ionizable cationic
lipids as described in International Patent Publication WO
2013/149140, which is incorporated herein by reference. In some
embodiments, the compositions and methods of the present invention
include a cationic lipid of one of the following formulas:
##STR00002##
or a pharmaceutically acceptable salt thereof, wherein R.sub.1 and
R.sub.2 are each independently selected from the group consisting
of hydrogen, an optionally substituted, variably saturated or
unsaturated C.sub.1-C.sub.20 alkyl and an optionally substituted,
variably saturated or unsaturated C.sub.6-C.sub.20 acyl; wherein
L.sub.1 and L.sub.2 are each independently selected from the group
consisting of hydrogen, an optionally substituted C.sub.1-C.sub.30
alkyl, an optionally substituted variably unsaturated
C.sub.1-C.sub.30 alkenyl, and an optionally substituted
C.sub.1-C.sub.30 alkynyl; wherein m and o are each independently
selected from the group consisting of zero and any positive integer
(e.g., where m is three); and wherein n is zero or any positive
integer (e.g., where n is one). In certain embodiments, the
compositions and methods of the present invention include the
cationic lipid (15Z,
18Z)--N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)
tetracosa-15,18-dien-1-amine ("HGT5000"), having a compound
structure of:
##STR00003##
and pharmaceutically acceptable salts thereof. In certain
embodiments, the compositions and methods of the present invention
include the cationic lipid (15Z,
18Z)--N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)
tetracosa-4,15,18-trien-1-amine ("HGT5001"), having a compound
structure of:
##STR00004##
and pharmaceutically acceptable salts thereof. In certain
embodiments, the compositions and methods of the present invention
include the cationic lipid and
(15Z,18Z)--N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)
tetracosa-5,15,18-trien-1-amine ("HGT5002"), having a compound
structure of:
##STR00005##
and pharmaceutically acceptable salts thereof.
[0167] Other suitable cationic lipids for use in the compositions
and methods of the invention include cationic lipids described as
aminoalcohol lipidoids in International Patent Publication WO
2010/053572, which is incorporated herein by reference. In certain
embodiments, the compositions and methods of the present invention
include a cationic lipid having a compound structure of:
##STR00006##
and pharmaceutically acceptable salts thereof.
[0168] Other suitable cationic lipids for use in the compositions
and methods of the invention include the cationic lipids as
described in International Patent Publication WO 2016/118725, which
is incorporated herein by reference. In certain embodiments, the
compositions and methods of the present invention include a
cationic lipid having a compound structure of:
##STR00007##
and pharmaceutically acceptable salts thereof.
[0169] Other suitable cationic lipids for use in the compositions
and methods of the invention include the cationic lipids as
described in International Patent Publication WO 2016/118724, which
is incorporated herein by reference. In certain embodiments, the
compositions and methods of the present invention include a
cationic lipid having a compound structure of:
##STR00008##
and pharmaceutically acceptable salts thereof.
[0170] Other suitable cationic lipids for use in the compositions
and methods of the invention include a cationic lipid having the
formula of 14,25-ditridecyl 15,18,21,24-tetraaza-octatriacontane,
and pharmaceutically acceptable salts thereof.
[0171] Other suitable cationic lipids for use in the compositions
and methods of the invention include the cationic lipids as
described in International Patent Publications WO 2013/063468 and
WO 2016/205691, each of which are incorporated herein by reference.
In some embodiments, the compositions and methods of the present
invention include a cationic lipid of the following formula:
##STR00009##
or pharmaceutically acceptable salts thereof, wherein each instance
of R.sup.L is independently optionally substituted C.sub.6-C.sub.40
alkenyl. In certain embodiments, the compositions and methods of
the present invention include a cationic lipid having a compound
structure of:
##STR00010##
and pharmaceutically acceptable salts thereof. In certain
embodiments, the compositions and methods of the present invention
include a cationic lipid having a compound structure of:
##STR00011##
and pharmaceutically acceptable salts thereof. In certain
embodiments, the compositions and methods of the present invention
include a cationic lipid having a compound structure of:
##STR00012##
and pharmaceutically acceptable salts thereof. In certain
embodiments, the compositions and methods of the present invention
include a cationic lipid having a compound structure of:
##STR00013##
and pharmaceutically acceptable salts thereof.
[0172] Other suitable cationic lipids for use in the compositions
and methods of the invention include the cationic lipids as
described in International Patent Publication WO 2015/184256, which
is incorporated herein by reference. In some embodiments, the
compositions and methods of the present invention include a
cationic lipid of the following formula:
##STR00014##
or a pharmaceutically acceptable salt thereof, wherein each X
independently is O or S; each Y independently is O or S; each m
independently is 0 to 20; each n independently is 1 to 6; each
R.sub.A is independently hydrogen, optionally substituted C1-50
alkyl, optionally substituted C2-50 alkenyl, optionally substituted
C2-50 alkynyl, optionally substituted C3-10 carbocyclyl, optionally
substituted 3-14 membered heterocyclyl, optionally substituted
C6-14 aryl, optionally substituted 5-14 membered heteroaryl or
halogen; and each R.sub.B is independently hydrogen, optionally
substituted C1-50 alkyl, optionally substituted C2-50 alkenyl,
optionally substituted C2-50 alkynyl, optionally substituted C3-10
carbocyclyl, optionally substituted 3-14 membered heterocyclyl,
optionally substituted C6-14 aryl, optionally substituted 5-14
membered heteroaryl or halogen. In certain embodiments, the
compositions and methods of the present invention include a
cationic lipid, "Target 23", having a compound structure of:
##STR00015##
and pharmaceutically acceptable salts thereof.
[0173] Other suitable cationic lipids for use in the compositions
and methods of the invention include the cationic lipids as
described in International Patent Publication WO 2016/004202, which
is incorporated herein by reference. In some embodiments, the
compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00016##
or a pharmaceutically acceptable salt thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00017##
or a pharmaceutically acceptable salt thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00018##
or a pharmaceutically acceptable salt thereof.
[0174] Other suitable cationic lipids for use in the compositions
and methods of the present invention include cationic lipids as
described in U.S. Provisional Patent Application Ser. No.
62/758,179, which is incorporated herein by reference. In some
embodiments, the compositions and methods of the present invention
include a cationic lipid of the following formula:
##STR00019##
or a pharmaceutically acceptable salt thereof, wherein each R.sup.1
and R.sup.2 is independently H or C.sub.1-C.sub.6 aliphatic; each m
is independently an integer having a value of 1 to 4; each A is
independently a covalent bond or arylene; each L.sup.1 is
independently an ester, thioester, disulfide, or anhydride group;
each L.sup.2 is independently C.sub.2-C.sub.10 aliphatic; each
X.sup.1 is independently H or OH; and each R.sup.3 is independently
C.sub.6-C.sub.20 aliphatic. In some embodiments, the compositions
and methods of the present invention include a cationic lipid of
the following formula:
##STR00020##
or a pharmaceutically acceptable salt thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid of the following formula:
##STR00021##
or a pharmaceutically acceptable salt thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid of the following formula:
##STR00022##
or a pharmaceutically acceptable salt thereof.
[0175] Other suitable cationic lipids for use in the compositions
and methods of the present invention include the cationic lipids as
described in J. McClellan, M. C. King, Cell 2010, 141, 210-217 and
in Whitehead et al., Nature Communications (2014) 5:4277, which is
incorporated herein by reference. In certain embodiments, the
cationic lipids of the compositions and methods of the present
invention include a cationic lipid having a compound structure
of:
##STR00023##
and pharmaceutically acceptable salts thereof.
[0176] Other suitable cationic lipids for use in the compositions
and methods of the invention include the cationic lipids as
described in International Patent Publication WO 2015/199952, which
is incorporated herein by reference. In some embodiments, the
compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00024##
and pharmaceutically acceptable salts thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00025##
and pharmaceutically acceptable salts thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00026##
and pharmaceutically acceptable salts thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00027##
and pharmaceutically acceptable salts thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00028##
and pharmaceutically acceptable salts thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00029##
and pharmaceutically acceptable salts thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00030##
and pharmaceutically acceptable salts thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00031##
and pharmaceutically acceptable salts thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00032##
and pharmaceutically acceptable salts thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00033##
and pharmaceutically acceptable salts thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00034##
and pharmaceutically acceptable salts thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00035##
and pharmaceutically acceptable salts thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00036##
and pharmaceutically acceptable salts thereof.
[0177] Other suitable cationic lipids for use in the compositions
and methods of the invention include the cationic lipids as
described in International Patent Publication WO 2017/004143, which
is incorporated herein by reference. In some embodiments, the
compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00037##
and pharmaceutically acceptable salts thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00038##
and pharmaceutically acceptable salts thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00039##
and pharmaceutically acceptable salts thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00040##
and pharmaceutically acceptable salts thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00041##
and pharmaceutically acceptable salts thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00042##
and pharmaceutically acceptable salts thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00043##
and pharmaceutically acceptable salts thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00044##
and pharmaceutically acceptable salts thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00045##
and pharmaceutically acceptable salts thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00046##
and pharmaceutically acceptable salts thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00047##
and pharmaceutically acceptable salts thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00048##
and pharmaceutically acceptable salts thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00049##
and pharmaceutically acceptable salts thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00050##
and pharmaceutically acceptable salts thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00051##
and pharmaceutically acceptable salts thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00052##
and pharmaceutically acceptable salts thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00053##
and pharmaceutically acceptable salts thereof.
[0178] Other suitable cationic lipids for use in the compositions
and methods of the invention include the cationic lipids as
described in International Patent Publication WO 2017/075531, which
is incorporated herein by reference. In some embodiments, the
compositions and methods of the present invention include a
cationic lipid of the following formula:
##STR00054##
or a pharmaceutically acceptable salt thereof, wherein one of L' or
L.sup.2 is --O(C.dbd.O)--, --(C.dbd.O)O--, --C(.dbd.O)--, --O--,
--S(O).sub.x, --S--S--, --C(.dbd.O)S--, --SC(.dbd.O)--,
--NR.sup.aC(.dbd.O)--, --C(.dbd.O)NR.sup.a--,
NR.sup.aC(--O)NR.sup.a--, --OC(.dbd.O)NR.sup.a--, or
--NR.sup.aC(.dbd.O)O--; and the other of L.sup.1 or L.sup.2 is
--O(C.dbd.O)--, --(C.dbd.O)O--, --C(.dbd.O)--, --O--, --S(O).sub.x,
--S--S--, --C(.dbd.O)S--, SC(.dbd.O)--, --NR.sup.aC(.dbd.O)--,
--C(.dbd.O)NR.sup.a--, --NR.sup.aC(.dbd.O)NR.sup.a--,
--OC(.dbd.O)NR.sup.a-- or --NR.sup.aC(.dbd.O)O-- or a direct bond;
G.sup.1 and G.sup.2 are each independently unsubstituted
C.sub.1-C.sub.12 alkylene or C.sub.1-C.sub.12 alkenylene; G.sup.3
is C.sub.1-C.sub.24 alkylene, C.sub.1-C.sub.24 alkenylene,
C.sub.3-C.sub.8 cycloalkylene, C.sub.3-C.sub.8 cycloalkenylene;
R.sup.a is H or C.sub.1-C.sub.12 alkyl; R.sup.1 and R.sup.2 are
each independently C.sub.6-C.sub.24 alkyl or C.sub.6-C.sub.24
alkenyl; R.sup.3 is H, OR.sup.5, CN, --C(.dbd.O)OR.sup.4,
--OC(.dbd.O)R.sup.4 or --NR.sup.5C(.dbd.O)R.sup.4; R.sup.4 is
C.sub.1-C.sub.12 alkyl; R.sup.5 is H or C.sub.1-C.sub.6 alkyl; and
x is 0, 1 or 2.
[0179] Other suitable cationic lipids for use in the compositions
and methods of the invention include the cationic lipids as
described in International Patent Publication WO 2017/117528, which
is incorporated herein by reference. In some embodiments, the
compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00055##
and pharmaceutically acceptable salts thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00056##
and pharmaceutically acceptable salts thereof. In some embodiments,
the compositions and methods of the present invention include a
cationic lipid having the compound structure:
##STR00057##
and pharmaceutically acceptable salts thereof.
[0180] Other suitable cationic lipids for use in the compositions
and methods of the invention include the cationic lipids as
described in International Patent Publication WO 2017/049245, which
is incorporated herein by reference. In some embodiments, the
cationic lipids of the compositions and methods of the present
invention include a compound of one of the following formulas:
##STR00058##
and pharmaceutically acceptable salts thereof. For any one of these
four formulas, R.sub.4 is independently selected from
--(CH.sub.2).sub.nQ and --(CH.sub.2).sub.nCHQR; Q is selected from
the group consisting of --OR, --OH, --O(CH.sub.2).sub.nN(R).sub.2,
--OC(O)R, --CX.sub.3, --CN, --N(R)C(O)R, --N(H)C(O)R,
--N(R)S(O).sub.2R, --N(H)S(O).sub.2R, --N(R)C(O)N(R).sub.2,
--N(H)C(O)N(R).sub.2, --N(H)C(O)N(H)(R), --N(R)C(S)N(R).sub.2,
--N(H)C(S)N(R).sub.2, --N(H)C(S)N(H)(R), and a heterocycle; and n
is 1, 2, or 3. In certain embodiments, the compositions and methods
of the present invention include a cationic lipid having a compound
structure of:
##STR00059##
and pharmaceutically acceptable salts thereof. In certain
embodiments, the compositions and methods of the present invention
include a cationic lipid having a compound structure of:
##STR00060##
and pharmaceutically acceptable salts thereof. In certain
embodiments, the compositions and methods of the present invention
include a cationic lipid having a compound structure of:
##STR00061##
and pharmaceutically acceptable salts thereof. In certain
embodiments, the compositions and methods of the present invention
include a cationic lipid having a compound structure of:
##STR00062##
and pharmaceutically acceptable salts thereof.
[0181] Other suitable cationic lipids for use in the compositions
and methods of the invention include the cationic lipids as
described in International Patent Publication WO 2017/173054 and WO
2015/095340, each of which is incorporated herein by reference. In
certain embodiments, the compositions and methods of the present
invention include a cationic lipid having a compound structure
of:
##STR00063##
and pharmaceutically acceptable salts thereof. In certain
embodiments, the compositions and methods of the present invention
include a cationic lipid having a compound structure of:
##STR00064##
and pharmaceutically acceptable salts thereof. In certain
embodiments, the compositions and methods of the present invention
include a cationic lipid having a compound structure of:
##STR00065##
and pharmaceutically acceptable salts thereof. In certain
embodiments, the compositions and methods of the present invention
include a cationic lipid having a compound structure of:
##STR00066##
and pharmaceutically acceptable salts thereof.
[0182] Other suitable cationic lipids for use in the compositions
and methods of the present invention include cleavable cationic
lipids as described in International Patent Publication WO
2012/170889, which is incorporated herein by reference. In some
embodiments, the compositions and methods of the present invention
include a cationic lipid of the following formula:
##STR00067##
wherein R.sub.1 is selected from the group consisting of imidazole,
guanidinium, amino, imine, enamine, an optionally-substituted alkyl
amino (e.g., an alkyl amino such as dimethylamino) and pyridyl;
wherein R.sub.2 is selected from the group consisting of one of the
following two formulas:
##STR00068##
and wherein R.sub.3 and R.sub.4 are each independently selected
from the group consisting of an optionally substituted, variably
saturated or unsaturated C.sub.6-C.sub.20 alkyl and an optionally
substituted, variably saturated or unsaturated C.sub.6-C.sub.20
acyl; and wherein n is zero or any positive integer (e.g., one,
two, three, four, five, six, seven, eight, nine, ten, eleven,
twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen,
nineteen, twenty or more). In certain embodiments, the compositions
and methods of the present invention include a cationic lipid,
"HGT4001", having a compound structure of:
##STR00069##
and pharmaceutically acceptable salts thereof. In certain
embodiments, the compositions and methods of the present invention
include a cationic lipid, "HGT4002", having a compound structure
of:
##STR00070##
and pharmaceutically acceptable salts thereof. In certain
embodiments, the compositions and methods of the present invention
include a cationic lipid, "HGT4003", having a compound structure
of:
##STR00071##
and pharmaceutically acceptable salts thereof. In certain
embodiments, the compositions and methods of the present invention
include a cationic lipid, "HGT4004", having a compound structure
of:
##STR00072##
and pharmaceutically acceptable salts thereof. In certain
embodiments, the compositions and methods of the present invention
include a cationic lipid "HGT4005", having a compound structure
of:
##STR00073##
and pharmaceutically acceptable salts thereof.
[0183] Other suitable cationic lipids for use in the compositions
and methods of the present invention include cleavable cationic
lipids as described in U.S. Provisional Application No. 62/672,194,
filed May 16, 2018, and incorporated herein by reference. In
certain embodiments, the compositions and methods of the present
invention include a cationic lipid that is any of general formulas
or any of structures (1a)-(21a) and (1b)-(21b) and (22)-(237)
described in U.S. Provisional Application No. 62/672,194. In
certain embodiments, the compositions and methods of the present
invention include a cationic lipid that has a structure according
to Formula (I'),
##STR00074##
wherein: [0184] R.sup.X is independently --H, or
-L.sup.5A-L.sup.5B-B'; [0185] each of L.sup.1, L.sup.2, and L.sup.3
is independently a covalent bond, --C(O)--, --C(O)O--, --C(O)S--,
or --C(O)NR.sup.L--; [0186] each L.sup.4A and L.sup.5A is
independently --C(O)--, --C(O)O--, or --C(O)NR.sup.L--; [0187] each
L.sup.4B and L.sup.5B is independently C.sub.1-C.sub.20 alkylene;
C.sub.2-C.sub.20 alkenylene; or C.sub.2-C.sub.20 alkynylene; [0188]
each B and B' is NR.sup.4R.sup.5 or a 5- to 10-membered
nitrogen-containing heteroaryl; [0189] each R.sup.1, R.sup.2, and
R.sup.3 is independently C.sub.6-C.sub.30 alkyl, C.sub.6-C.sub.30
alkenyl, or C.sub.6-C.sub.30 alkynyl; [0190] each R.sup.4 and
R.sup.5 is independently hydrogen, C.sub.1-C.sub.10 alkyl;
C.sub.2-C.sub.10 alkenyl; or C.sub.2-C.sub.10 alkynyl; and [0191]
each R.sup.L is independently hydrogen, C.sub.1-C.sub.20 alkyl,
C.sub.2-C.sub.20 alkenyl, or C.sub.2-C.sub.20 alkynyl. In certain
embodiments, the compositions and methods of the present invention
include a cationic lipid that is Compound (139) of 62/672,194,
having a compound structure of:
##STR00075##
[0192] In some embodiments, the compositions and methods of the
present invention include the cationic lipid,
N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
("DOTMA"). (Feigner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987);
U.S. Pat. No. 4,897,355, which is incorporated herein by
reference). Other cationic lipids suitable for the compositions and
methods of the present invention include, for example,
5-carboxyspermylglycinedioctadecylamide ("DOGS");
2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanamin-
ium ("DOSPA") (Behr et al. Proc. Nat.'l Acad. Sci. 86, 6982 (1989),
U.S. Pat. Nos. 5,171,678; 5,334,761);
1,2-Dioleoyl-3-Dimethylammonium-Propane ("DODAP");
1,2-Dioleoyl-3-Trimethylammonium-Propane ("DOTAP").
[0193] Additional exemplary cationic lipids suitable for the
compositions and methods of the present invention also include:
1,2-distearyloxy-N,N-dimethyl-3-aminopropane ("DSDMA");
1,2-dioleyloxy-N,N-dimethyl-3-aminopropane ("DODMA");
1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane ("DLinDMA");
1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane ("DLenDMA");
N-dioleyl-N,N-dimethylammonium chloride ("DODAC");
N,N-distearyl-N,N-dimethylammonium bromide ("DDAB");
N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium
bromide ("DMRIE");
3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-
tadecadienoxy)propane ("CLinDMA");
2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',
1-2'-octadecadienoxy)propane ("CpLinDMA");
N,N-dimethyl-3,4-dioleyloxybenzylamine ("DMOBA");
1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane ("DOcarbDAP");
2,3-Dilinoleoyloxy-N,N-dimethylpropylamine ("DLinDAP");
1,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane ("DLincarbDAP");
1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane ("DLinCDAP");
2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane
("DLin-K-DMA"); 2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N,
N-dimethyl-3-[(9Z, 12Z)-octadeca-9, 12-dien-1-yloxy]propane-1-amine
("Octyl-CLinDMA");
(2R)-2-((8-[(3beta)-cholest-5-en-3-yloxy]octyl)oxy)-N,
N-dimethyl-3-[(9Z, 12Z)-octadeca-9, 12-dien-1-yloxy]propan-1-amine
("Octyl-CLinDMA (2R)");
(2S)-2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N,
fsl-dimethyh3-[(9Z, 12Z)-octadeca-9, 12-dien-1-yloxy]propan-1-amine
("Octyl-CLinDMA (2S)");
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane
("DLin-K-XTC2-DMA"); and 2-(2,2-di((9Z,12Z)-octadeca-9,1
2-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine
("DLin-KC2-DMA") (see, WO 2010/042877, which is incorporated herein
by reference; Semple et al., Nature Biotech. 28: 172-176 (2010)).
(Heyes, J., et al., J Controlled Release 107: 276-287 (2005);
Morrissey, D V., et al., Nat. Biotechnol. 23(8): 1003-1007 (2005);
International Patent Publication WO 2005/121348). In some
embodiments, one or more of the cationic lipids comprise at least
one of an imidazole, dialkylamino, or guanidinium moiety.
[0194] In some embodiments, one or more cationic lipids suitable
for the compositions and methods of the present invention include
2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane ("XTC");
(3aR,5s,6aS)--N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydr-
o-3aH-cyclopenta[d][1,3]dioxol-5-amine ("ALNY-100") and/or
4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetr-
aazahexadecane-1,16-diamide ("NC98-5").
[0195] In some embodiments, the compositions of the present
invention include one or more cationic lipids that constitute at
least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
or 70%, measured by weight, of the total lipid content in the
composition, e.g., a lipid nanoparticle. In some embodiments, the
compositions of the present invention include one or more cationic
lipids that constitute at least about 5%, 10%, 20%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, or 70%, measured as a mol %, of the total
lipid content in the composition, e.g., a lipid nanoparticle. In
some embodiments, the compositions of the present invention include
one or more cationic lipids that constitute about 30-70% (e.g.,
about 30-65%, about 30-60%, about 30-55%, about 30-50%, about
30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%),
measured by weight, of the total lipid content in the composition,
e.g., a lipid nanoparticle. In some embodiments, the compositions
of the present invention include one or more cationic lipids that
constitute about 30-70% (e.g., about 30-65%, about 30-60%, about
30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%,
about 35-45%, or about 35-40%), measured as mol %, of the total
lipid content in the composition, e.g., a lipid nanoparticle.
[0196] Non-Cationic/Helper Lipids
[0197] In some embodiments, a suitable lipid solution includes one
or more non-cation/helper lipids. As used herein, the phrase
"non-cationic lipid" refers to any neutral, zwitterionic or anionic
lipid. As used herein, the phrase "anionic lipid" refers to any of
a number of lipid species that carry a net negative charge at a
selected H, such as physiological pH. Non-cationic lipids include,
but are not limited to, distearoylphosphatidylcholine (DSPC),
dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine
(DPPC), dioleoylphosphatidylglycerol (DOPG),
dipalmitoylphosphatidylglycerol (DPPG),
dioleoylphosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoyl-phosphatidylethanolamine (POPE),
dioleoyl-phosphatidylethanolamine
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),
dipalmitoyl phosphatidyl ethanolamine (DPPE),
dimyristoylphosphoethanolamine (DMPE), di
stearoyl-phosphatidyl-ethanolamine (DSPE), phosphatidylserine,
sphingolipids, cerebrosides, gangliosides, 16-O-monomethyl PE,
16-O-dimethyl PE, 18-1-trans PE,
1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), or a mixture
thereof.
[0198] In some embodiments, such non-cationic lipids may be used
alone, but are preferably used in combination with other lipids,
for example, cationic lipids. In some embodiments, the non-cationic
lipid may comprise a molar ratio of about 5% to about 90%, or about
10% to about 70% of the total lipid present in a liposome. In some
embodiments, a non-cationic lipid is a neutral lipid, i.e., a lipid
that does not carry a net charge in the conditions under which the
composition is formulated and/or administered. In some embodiments,
the percentage of non-cationic lipid in a liposome may be greater
than 5%, greater than 10%, greater than 20%, greater than 30%, or
greater than 40%.
[0199] In some embodiments, non-cationic lipids may constitute at
least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65% or 70% of the total lipids in a suitable lipid solution by
weight or by molar. In some embodiments, non-cationic lipid(s)
constitute(s) about 30-50% (e.g., about 30-45%, about 30-40%, about
35-50%, about 35-45%, or about 35-40%) of the total lipids in a
suitable lipid solution by weight or by molar.
[0200] In some embodiments, one or more non-cationic lipids are
used in the process described herein. In some embodiments, the one
or more non-cationic lipids comprise a non-cationic lipid selected
from the group consisting of DSPC
(1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC
(1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE
(1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC
(1,2-dioleyl-sn-glycero-3-phosphotidylcholine) DPPE
(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE
(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG
(2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)), and
combinations thereof
[0201] Cholesterol-Based Lipids
[0202] In some embodiments, a suitable lipid solution includes one
or more cholesterol-based lipids. For example, suitable
cholesterol-based cationic lipids include, for example, DC-Choi
(N,N-dimethyl-N-ethylcarboxamidocholesterol),1,4-bis(3-N-oleylamino-propy-
l)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280
(1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Pat. No.
5,744,335), or ICE. In some embodiments, the cholesterol-based
lipid may comprise a molar ration of about 2% to about 30%, or
about 5% to about 20% of the total lipid present in a liposome. In
some embodiments, the percentage of cholesterol-based lipid in the
lipid nanoparticle may be greater than 5%, greater than 10%,
greater than 20%, greater than 30%, or greater than 40%.
[0203] In some embodiments, cholesterol-based lipid(s)
constitute(s) at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, or
70% of the total lipids in a suitable lipid solution by weight or
by molar. In some embodiments, cholesterol-based lipid(s)
constitute(s) about 30-50% (e.g., about 30-45%, about 30-40%, about
35-50%, about 35-45%, or about 35-40%) of the total lipids in a
suitable lipid solution by weight or by molar.
[0204] PEGylated Lipids
[0205] In some embodiments, a suitable lipid solution includes one
or more PEGylated lipids. The use of polyethylene glycol
(PEG)-modified phospholipids and derivatized lipids such as
derivatized ceramides (PEG-CER), including
N-Octanoyl-Sphingosine-1-[Succinyl(Methoxy Polyethylene
Glycol)-2000](C.sub.8 PEG-2000 ceramide) is also contemplated by
the present invention, either alone or preferably in combination
with other lipid formulations together which comprise the transfer
vehicle (e.g., a lipid nanoparticle). Contemplated PEG-modified
lipids include, but are not limited to, a polyethylene glycol chain
of up to 5 kDa in length covalently attached to a lipid with alkyl
chain(s) of C.sub.6-C.sub.20 length. The addition of such
components may prevent complex aggregation and may also provide a
means for increasing circulation lifetime and increasing the
delivery of the lipid-nucleic acid composition to the target
tissues, (Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237),
or they may be selected to rapidly exchange out of the formulation
in vivo (see U.S. Pat. No. 5,885,613). Particularly useful
exchangeable lipids are PEG-ceramides having shorter acyl chains
(e.g., C.sub.14 or C.sub.18). The PEG-modified phospholipid and
derivitized lipids of the present invention may comprise a molar
ratio from about 0% to about 20%, about 0.5% to about 20%, about 1%
to about 15%, about 4% to about 10%, or about 2% of the total lipid
present in the liposomal transfer vehicle.
[0206] According to various embodiments, the selection of cationic
lipids, non-cationic lipids and/or PEG-modified lipids which
comprise the lipid nanoparticle, as well as the relative molar
ratio of such lipids to each other, is based upon the
characteristics of the selected lipid(s), the nature of the
intended target cells, the characteristics of the mRNA to be
delivered. Additional considerations include, for example, the
saturation of the alkyl chain, as well as the size, charge, pH,
pKa, fusogenicity and toxicity of the selected lipid(s). Thus the
molar ratios may be adjusted accordingly.
[0207] In some embodiments, a suitable delivery vehicle is
formulated using a polymer as a carrier, alone or in combination
with other carriers including various lipids described herein.
Thus, in some embodiments, liposomal delivery vehicles, as used
herein, also encompass nanoparticles comprising polymers. Suitable
polymers may include, for example, polyacrylates,
polyalkycyanoacrylates, polylactide, polylactide-polyglycolide
copolymers, polycaprolactones, dextran, albumin, gelatin, alginate,
collagen, chitosan, cyclodextrins, protamine, PEGylated protamine,
PLL, PEGylated PLL and polyethylenimine (PEI). When PEI is present,
it may be branched PEI of a molecular weight ranging from 10 to 40
kDa, e.g., 25 kDa branched PEI (Sigma #408727).
[0208] A suitable liposome for the present invention may include
one or more of any of the cationic lipids, non-cationic lipids,
cholesterol lipids, PEG-modified lipids and/or polymers described
herein at various ratios. As non-limiting examples, a suitable
liposome formulation may include a combination selected from
cKK-E12, DOPE, cholesterol and DMG-PEG2K; C.sub.12-200, DOPE,
cholesterol and DMG-PEG2K; HGT4003, DOPE, cholesterol and
DMG-PEG2K; ICE, DOPE, cholesterol and DMG-PEG2K; or ICE, DOPE, and
DMG-PEG2K.
[0209] In various embodiments, cationic lipids (e.g., cKK-E12,
C.sub.12-200, ICE, and/or HGT4003) constitute about 30-60% (e.g.,
about 30-55%, about 30-50%, about 30-45%, about 30-40%, about
35-50%, about 35-45%, or about 35-40%) of the liposome by molar
ratio. In some embodiments, the percentage of cationic lipids
(e.g., cKK-E12, C.sub.12-200, ICE, and/or HGT4003) is or greater
than about 30%, about 35%, about 40%, about 45%, about 50%, about
55%, or about 60% of the liposome by molar ratio.
[0210] In some embodiments, the ratio of cationic lipid(s) to
non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified
lipid(s) may be between about 30-60:25-35:20-30:1-15, respectively.
In some embodiments, the ratio of cationic lipid(s) to non-cationic
lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is
approximately 40:30:20:10, respectively. In some embodiments, the
ratio of cationic lipid(s) to non-cationic lipid(s) to
cholesterol-based lipid(s) to PEG-modified lipid(s) is
approximately 40:30:25:5, respectively. In some embodiments, the
ratio of cationic lipid(s) to non-cationic lipid(s) to
cholesterol-based lipid(s) to PEG-modified lipid(s) is
approximately 40:32:25:3, respectively. In some embodiments, the
ratio of cationic lipid(s) to non-cationic lipid(s) to
cholesterol-based lipid(s) to PEG-modified lipid(s) is
approximately 50:25:20:5. In some embodiments, the ratio of sterol
lipid(s) to non-cationic lipid(s) to PEG-modified lipid(s) is
50:45:5. In some embodiments, the ratio of sterol lipid(s) to
non-cationic lipid(s) to PEG-modified lipid(s) is 50:40:10. In some
embodiments, the ratio of sterol lipid(s) to non-cationic lipid(s)
to PEG-modified lipid(s) is 55:40:5. In some embodiments, the ratio
of sterol lipid(s) to non-cationic lipid(s) to PEG-modified
lipid(s) is 55:35:10. In some embodiments, the ratio of sterol
lipid(s) to non-cationic lipid(s) to PEG-modified lipid(s) is
60:35:5. In some embodiments, the ratio of sterol lipid(s) to
non-cationic lipid(s) to PEG-modified lipid(s) is 60:30:10.
[0211] In some embodiments, a suitable liposome for the present
invention comprises ICE and DOPE at an ICE:DOPE molar ratio of
>1:1. In some embodiments, the ICE:DOPE molar ratio is
<2.5:1. In some embodiments, the ICE:DOPE molar ratio is between
1:1 and 2.5:1. In some embodiments, the ICE:DOPE molar ratio is
approximately 1.5:1. In some embodiments, the ICE:DOPE molar ratio
is approximately 1.7:1. In some embodiments, the ICE:DOPE molar
ratio is approximately 2:1. In some embodiments, a suitable
liposome for the present invention comprises ICE and DMG-PEG-2K at
an ICE:DMG-PEG-2K molar ratio of >10:1. In some embodiments, the
ICE:DMG-PEG-2K molar ratio is <16:1. In some embodiments, the
ICE:DMG-PEG-2K molar ratio is approximately 12:1. In some
embodiments, the ICE:DMG-PEG-2K molar ratio is approximately 14:1.
In some embodiments, a suitable liposome for the present invention
comprises DOPE and DMG-PEG-2K at a DOPE:DMG-PEG-2K molar ratio of
>5:1. In some embodiments, the DOPE:DMG-PEG-2K molar ratio is
<11:1. In some embodiments, the DOPE:DMG-PEG-2K molar ratio is
approximately 7:1. In some embodiments, the DOPE:DMG-PEG-2K molar
ratio is approximately 10:1. In some embodiments, a suitable
liposome for the present invention comprises ICE, DOPE and
DMG-PEG-2K at an ICE:DOPE:DMG-PEG-2K molar ratio of 50:45:5. In
some embodiments, a suitable liposome for the present invention
comprises ICE, DOPE and DMG-PEG-2K at an ICE:DOPE:DMG-PEG-2K molar
ratio of 50:40:10. In some embodiments, a suitable liposome for the
present invention comprises ICE, DOPE and DMG-PEG-2K at an
ICE:DOPE:DMG-PEG-2K molar ratio of 55:40:5. In some embodiments, a
suitable liposome for the present invention comprises ICE, DOPE and
DMG-PEG-2K at an ICE:DOPE:DMG-PEG-2K molar ratio of 55:35:10. In
some embodiments, a suitable liposome for the present invention
comprises ICE, DOPE and DMG-PEG-2K at an ICE:DOPE:DMG-PEG-2K molar
ratio of 60:35:5. In some embodiments, a suitable liposome for the
present invention comprises ICE, DOPE and DMG-PEG-2K at an
ICE:DOPE:DMG-PEG-2K molar ratio of 60:30:10.
[0212] In some embodiments, the use of polyethylene glycol
(PEG)-modified phospholipids and derivatized lipids such as
derivatized ceramides (PEG-CER), including
N-Octanoyl-Sphingosine-1-[Succinyl(Methoxy Polyethylene
Glycol)-2000] (C8 PEG-2000 ceramide) is also contemplated by the
present invention. Contemplated PEG-modified lipids include, but
are not limited to, a polyethylene glycol chain of up to 2 kDa, up
to 3 kDa, up to 4 kDa or up to 5 kDa in length covalently attached
to a lipid with alkyl chain(s) of C.sub.6-C.sub.20 length. In some
embodiments, a PEG-modified or PEGylated lipid is PEGylated
cholesterol or PEG-2K. In some embodiments, particularly useful
exchangeable lipids are PEG-ceramides having shorter acyl chains
(e.g., C.sub.14 or C.sub.18).
[0213] PEG-modified phospholipid and derivatized lipids may
constitute no greater than about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%,
3.5%, 4%, 4.5% or 5% of the total lipids in a suitable lipid
solution by weight or by molar. In some embodiments, PEG-modified
lipids may constitute about 5% or less of the total lipids in a
suitable lipid solution by weight or by molar concentration. In
some embodiments, PEG-modified lipids may constitute about 4% or
less of the total lipids in a suitable lipid solution by weight or
by molar concentration. In some embodiments, PEG-modified lipids
typically constitute 3% or less of total lipids in a suitable lipid
solution by weight or by molar concentration. In some embodiments,
PEG-modified lipids typically constitute 2% or less of total lipids
in a suitable lipid solution by weight or by molar concentration.
In some embodiments, PEG-modified lipids typically constitute 1% or
less of total lipids in a suitable lipid solution by weight or by
molar concentration. In some embodiments, PEG-modified lipids
constitute about 1-5%, about 1-4%, about 1-3%, or about 1-2%) of
the total lipids in a suitable lipid solution by weight or by molar
concentration. In some embodiments, PEG modified lipids constitute
about 0.01-3% (e.g., about 0.01-2.5%, 0.01-2%, 0.01-1.5%, 0.01-1%)
of the total lipids in a suitable lipid solution by weight or by
molar concentration.
[0214] Various combinations of lipids, i.e., cationic lipids,
non-cationic lipids, PEG-modified lipids and optionally
cholesterol, that can used to prepare, and that are comprised in,
preformed lipid nanoparticles are described in the literature and
herein. For example, a suitable lipid solution may contain cKK-E12,
DOPE, cholesterol, and DMG-PEG2K; C.sub.12-200, DOPE, cholesterol,
and DMG-PEG2K; HGT5000, DOPE, cholesterol, and DMG-PEG2K; HGT5001,
DOPE, cholesterol, and DMG-PEG2K; cKK-E12, DPPC, cholesterol, and
DMG-PEG2K; C.sub.12-200, DPPC, cholesterol, and DMG-PEG2K; HGT5000,
DPPC, cholesterol, and DMG-PEG2K; HGT5001, DPPC, cholesterol, and
DMG-PEG2K; or ICE, DOPE and DMG-PEG2K. Additional combinations of
lipids are described in the art, e.g., PCT/US17/61100, filed on
Nov. 10, 2017, published as WO 2018/089790; entitled "Novel
ICE-based Lipid Nanoparticle Formulation for Delivery of mRNA,";
PCT/US18/21292, filed on Mar. 7, 2018, published as WO 2018/165257,
entitled "PolyAnionic Delivery of Nucleic Acids"; PCT/US18/36920,
filed on Jun. 11, 2018, entitled, "Poly (Phosphoesters) for
Delivery of Nucleic Acids." The selection of cationic lipids,
non-cationic lipids and/or PEG-modified lipids which comprise the
lipid mixture as well as the relative molar ratio of such lipids to
each other, is based upon the characteristics of the selected
lipid(s) and the nature of the and the characteristics of the mRNA
to be encapsulated. Additional considerations include, for example,
the saturation of the alkyl chain, as well as the size, charge, pH,
pKa, fusogenicity and toxicity of the selected lipid(s). Thus the
molar ratios may be adjusted accordingly.
[0215] In various embodiments, cationic lipids (e.g., cKK-E12,
C12-200, ICE, and/or HGT4003) constitute about 30-60% (e.g., about
30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%,
about 35-45%, or about 35-40%) of the liposome by molar ratio. In
some embodiments, the percentage of cationic lipids (e.g., cKK-E12,
C12-200, ICE, and/or HGT4003) is or greater than about 30%, about
35%, about 40%, about 45%, about 50%, about 55%, or about 60% of
the liposome by molar ratio.
Provided Nanoparticles Encapsulating mRNA
[0216] A process according to the present invention results in more
homogeneous and smaller particle sizes (e.g., particle sizes of
about 75-150 nm (e.g. 75, 80, 85, 90, 95, 100, 105, 110, 115, 120,
125, 130, 135, 140, 145, or 150 nm) as well as significantly
improved encapsulation efficiency and/or mRNA recovery rate as
compared to a prior art process.
[0217] Thus, the present invention provides a composition
comprising purified nanoparticles described herein. In some
embodiments, a majority of purified nanoparticles in a composition,
i.e., greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 96%, 97%, 98%, or 99% of the purified nanoparticles, have
a size less than about 100 nm (e.g., less than about 95 nm, about
90 nm, about 85 nm, about 80 nm, about 75 nm, about 70 nm, about 65
nm, about 60 nm, about 55 nm, or about 50 nm). In some embodiments,
substantially all of the purified nanoparticles have a size less
than 100 nm (e.g., less than about 95 nm, about 90 nm, about 85 nm,
about 80 nm, about 75 nm, about 70 nm, about 65 nm, about 60 nm,
about 55 nm, or about 50 nm).
[0218] In addition, more homogeneous nanoparticles with narrow
particle size range are achieved by a process of the present
invention. For example, greater than about 70%, 75%, 80%, 85%, 90%,
95%, 96%, 97%, 98%, 99% of the purified nanoparticles in a
composition provided by the present invention have a size ranging
from about 40-90 nm (e.g., about 40-85 nm, about 40-80 nm, about
40-75 nm, about 40-70 nm, about 40-65 nm, or about 40-60 nm). In
some embodiments, substantially all of the purified nanoparticles
have a size ranging from about 40-90 nm (e.g., about 40-85 nm,
about 40-80 nm, about 40-75 nm, about 40-70 nm, about 40-65 nm, or
about 40-60 nm).
[0219] In some embodiments, the dispersity, or measure of
heterogeneity in size of molecules (PDI), of nanoparticles in a
composition provided by the present invention is less than about
0.3 (e.g., less than about 0.3, 0.2, 0.15, 0.14, 0.13, 0.12, 0.11,
0.10, 0.09, or 0.08).
[0220] In some embodiments, greater than about 75%, 80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% of the purified lipid nanoparticles in a
composition provided by the present invention encapsulate an mRNA
within each individual particle. In some embodiments, substantially
all of the purified lipid nanoparticles in a composition
encapsulate an mRNA within each individual particle.
[0221] In some embodiments, a composition according to the present
invention contains at least about 1 mg, 5 mg, 10 mg, 100 mg, 500
mg, or 1000 mg of encapsulated mRNA. In some embodiments, a process
according to the present invention results in greater than about
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%
recovery of mRNA.
[0222] This invention is further illustrated by the following
examples that should not be construed as limiting. The contents of
all references, patents, and published patent applications cited
throughout this application are herein incorporated by reference in
their entirety for all purposes.
Purification
[0223] In some embodiments, the encapsulated nucleic acids (e.g.
encapsulated mRNA) in lipid nanoparticles are further purified
and/or concentrated. Various purification methods may be used. In
some embodiments, lipid nanoparticles are purified using Tangential
Flow Filtration. Tangential flow filtration (TFF), also referred to
as cross-flow filtration, is a type of filtration wherein the
material to be filtered is passed tangentially across a filter
rather than through it. In TFF, undesired permeate passes through
the filter, while the desired retentate passes along the filter and
is collected downstream. It is important to note that the desired
material is typically contained in the retentate in TFF, which is
the opposite of what one normally encounters in traditional-dead
end filtration.
[0224] Depending upon the material to be filtered, TFF is usually
used for either microfiltration or ultrafiltration. Microfiltration
is typically defined as instances where the filter has a pore size
of between 0.05 .mu.m and 1.0 .mu.m, inclusive, while
ultrafiltration typically involves filters with a pore size of less
than 0.05 .mu.m. Pore size also determines the nominal molecular
weight limits (NMWL), also referred to as the molecular weight cut
off (MWCO) for a particular filter, with microfiltration membranes
typically having NMWLs of greater than 1,000 kilodaltons (kDa) and
ultrafiltration filters having NMWLs of between 1 kDa and 1,000
kDa.
[0225] A principal advantage of tangential flow filtration is that
non-permeable particles that may aggregate in and block the filter
(sometimes referred to as "filter cake") during traditional
"dead-end" filtration, are instead carried along the surface of the
filter. This advantage allows tangential flow filtration to be
widely used in industrial processes requiring continuous operation
since down time is significantly reduced because filters do not
generally need to be removed and cleaned.
[0226] Tangential flow filtration can be used for several purposes
including concentration and diafiltration, among others.
Concentration is a process whereby solvent is removed from a
solution while solute molecules are retained. In order to
effectively concentrate a sample, a membrane having a NMWL or MWCO
that is substantially lower than the molecular weight of the solute
molecules to be retained is used. Generally, one of skill may
select a filter having a NMWL or MWCO of three to six times below
the molecular weight of the target molecule(s).
[0227] Diafiltration is a fractionation process whereby small
undesired particles are passed through a filter while larger
desired nanoparticles are maintained in the retentate without
changing the concentration of those nanoparticles in solution.
Diafiltration is often used to remove salts or reaction buffers
from a solution. Diafiltration may be either continuous or
discontinuous. In continuous diafiltration, a diafiltration
solution is added to the sample feed at the same rate that filtrate
is generated. In discontinuous diafiltration, the solution is first
diluted and then concentrated back to the starting concentration.
Discontinuous diafiltration may be repeated until a desired
concentration of nanoparticles is reached.
[0228] In some embodiments, purification and/or concentration steps
include dialysis, gel filtration, centrifugation (e.g. use of
Amicon centrifugal filters), vacuum, and pressure-press (e.g.
French press).
[0229] Purified and/or concentrated lipid nanoparticles may be
formulated in a desired buffer such as, for example, PBS.
Therapeutic Indications
[0230] In some embodiments, the present invention can be used to
encapsulate mRNA in lipids, thus producing lipid encapsulated mRNA
for use in the treatment of various disorders, diseases, conditions
and/or syndromes. Non-limiting examples of select uses of the
invention as described herein are described in the paragraphs that
follow.
[0231] In some embodiments, the present invention provides a method
for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes a peptide or polypeptide for use in
the delivery to or treatment of a human subject. In some
embodiments, therapeutic composition comprising lipid encapsulated
mRNA is used for delivery in the lung of a subject or a lung cell.
In certain embodiments, the present invention provides a method for
producing a therapeutic composition comprising lipid encapsulated
mRNA that encodes an endogenous protein which may be deficient or
non-functional in a subject. In certain embodiments, the present
invention provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes an endogenous
protein which may be deficient or non-functional in a subject.
[0232] In certain embodiments, the present invention provides a
method for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes a peptide or polypeptide for use in
the delivery to or treatment of the lung of a subject or a lung
cell. In certain embodiments, the present invention is useful in a
method for manufacturing mRNA encoding cystic fibrosis
transmembrane conductance regulator, CFTR. The CFTR mRNA is
delivered to the lung of a subject in need in a therapeutic
composition for treating cystic fibrosis. In certain embodiments,
the present invention provides a method for producing a therapeutic
composition comprising lipid encapsulated mRNA that encodes a
peptide or polypeptide for use in the delivery to or treatment of
the liver of a subject or a liver cell. Such peptides and
polypeptides can include those associated with a urea cycle
disorder, associated with a lysosomal storage disorder, with a
glycogen storage disorder, associated with an amino acid metabolism
disorder, associated with a lipid metabolism or fibrotic disorder,
associated with methyl malonic acidemia, or associated with any
other metabolic disorder for which delivery to or treatment of the
liver or a liver cell with enriched full-length mRNA provides
therapeutic benefit.
[0233] In certain embodiments, the present invention provides a
method for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes for a protein associated with a urea
cycle disorder. In certain embodiments, the present invention
provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for ornithine
transcarbamylase (OTC) protein. In certain embodiments, the present
invention provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for arginosuccinate
synthetase 1 protein. In certain embodiments, the present invention
provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for carbamoyl
phosphate synthetase I protein. In certain embodiments, the present
invention provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for arginosuccinate
lyase protein. In certain embodiments, the present invention
provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for arginase
protein.
[0234] In certain embodiments, the present invention provides a
method for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes for a protein associated with a
lysosomal storage disorder. In certain embodiments, the present
invention provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for alpha
galactosidase protein. In certain embodiments, the present
invention provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for
glucocerebrosidase protein. In certain embodiments, the present
invention provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for
iduronate-2-sulfatase protein. In certain embodiments, the present
invention provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for iduronidase
protein. In certain embodiments, the present invention provides a
method for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes for N-acetyl-alpha-D-glucosaminidase
protein. In certain embodiments, the present invention provides a
method for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes for heparan N-sulfatase protein. In
certain embodiments, the present invention provides a method for
producing a therapeutic composition comprising lipid encapsulated
mRNA that encodes for galactosamine-6 sulfatase protein. In certain
embodiments, the present invention provides a method for producing
a therapeutic composition comprising lipid encapsulated mRNA that
encodes for beta-galactosidase protein. In certain embodiments, the
present invention provides a method for producing a therapeutic
composition comprising lipid encapsulated mRNA that encodes for
lysosomal lipase protein. In certain embodiments, the present
invention provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for arylsulfatase B
(N-acetylgalactosamine-4-sulfatase) protein. In certain
embodiments, the present invention provides a method for producing
a therapeutic composition comprising lipid encapsulated mRNA that
encodes for transcription factor EB (TFEB).
[0235] In certain embodiments, the present invention provides a
method for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes for a protein associated with a
glycogen storage disorder. In certain embodiments, the present
invention provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for acid
alpha-glucosidase protein. In certain embodiments, the present
invention provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for
glucose-6-phosphatase (G6PC) protein. In certain embodiments, the
present invention provides a method for producing a therapeutic
composition comprising lipid encapsulated mRNA that encodes for
liver glycogen phosphorylase protein. In certain embodiments, the
present invention provides a method for producing a therapeutic
composition comprising lipid encapsulated mRNA that encodes for
muscle phosphoglycerate mutase protein. In certain embodiments, the
present invention provides a method for producing a therapeutic
composition comprising lipid encapsulated mRNA that encodes for
glycogen debranching enzyme.
[0236] In certain embodiments, the present invention provides a
method for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes for a protein associated with amino
acid metabolism. In certain embodiments, the present invention
provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for phenylalanine
hydroxylase enzyme. In certain embodiments, the present invention
provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for glutaryl-CoA
dehydrogenase enzyme. In certain embodiments, the present invention
provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for propionyl-CoA
caboxylase enzyme. In certain embodiments, the present invention
provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for oxalase
alanine-glyoxylate aminotransferase enzyme.
[0237] In certain embodiments, the present invention provides a
method for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes for a protein associated with a
lipid metabolism or fibrotic disorder. In certain embodiments, the
present invention provides a method for producing a therapeutic
composition comprising lipid encapsulated mRNA that encodes for an
mTOR inhibitor. In certain embodiments, the present invention
provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for ATPase
phospholipid transporting 8B1 (ATP8B1) protein. In certain
embodiments, the present invention provides a method for producing
a therapeutic composition comprising lipid encapsulated mRNA that
encodes for one or more NF-kappa B inhibitors, such as one or more
of I-kappa B alpha, interferon-related development regulator 1
(IFRD1), and Sirtuin 1 (SIRT1). In certain embodiments, the present
invention provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for PPAR-gamma
protein or an active variant.
[0238] In certain embodiments, the present invention provides a
method for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes for a protein associated with methyl
malonic acidemia. For example, in certain embodiments, the present
invention provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for methyl malonyl
CoA mutase protein. In certain embodiments, the present invention
provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for methylmalonyl
CoA epimerase protein.
[0239] In certain embodiments, the present invention provides a
method for producing a therapeutic composition comprising lipid
encapsulated mRNA for which delivery to or treatment of the liver
can provide therapeutic benefit. In certain embodiments, the
present invention provides a method for producing a therapeutic
composition comprising lipid encapsulated mRNA that encodes for
ATP7B protein, also known as Wilson disease protein. In certain
embodiments, the present invention provides a method for producing
a therapeutic composition comprising lipid encapsulated mRNA that
encodes for porphobilinogen deaminase enzyme. In certain
embodiments, the present invention provides a method for producing
a therapeutic composition comprising lipid encapsulated mRNA that
encodes for one or clotting enzymes, such as Factor VIII, Factor
IX, Factor VII, and Factor X. In certain embodiments, the present
invention provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for human
hemochromatosis (HFE) protein.
[0240] In certain embodiments, the present invention provides a
method for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes a peptide or polypeptide for use in
the delivery to or treatment of the cardiovascular conditions of a
subject or a cardiovascular cell. In certain embodiments, the
present invention provides a method for producing a therapeutic
composition comprising lipid encapsulated mRNA that encodes for
vascular endothelial growth factor A protein. In certain
embodiments, the present invention provides a method for producing
a therapeutic composition comprising lipid encapsulated mRNA that
encodes for relaxin protein. In certain embodiments, the present
invention provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for bone
morphogenetic protein-9 protein. In certain embodiments, the
present invention provides a method for producing a therapeutic
composition comprising lipid encapsulated mRNA that encodes for
bone morphogenetic protein-2 receptor protein.
[0241] In certain embodiments, the present invention provides a
method for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes a peptide or polypeptide for use in
the delivery to or treatment of the muscle of a subject or a muscle
cell. In certain embodiments, the present invention provides a
method for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes for dystrophin protein. In certain
embodiments, the present invention provides a method for producing
a therapeutic composition comprising lipid encapsulated mRNA that
encodes for frataxin protein. In certain embodiments, the present
invention provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes a peptide or
polypeptide for use in the delivery to or treatment of the cardiac
muscle of a subject or a cardiac muscle cell. In certain
embodiments, the present invention provides a method for producing
a therapeutic composition comprising lipid encapsulated mRNA that
encodes for a protein that modulates one or both of a potassium
channel and a sodium channel in muscle tissue or in a muscle cell.
In certain embodiments, the present invention provides a method for
producing a therapeutic composition comprising lipid encapsulated
mRNA that encodes for a protein that modulates a Kv7.1 channel in
muscle tissue or in a muscle cell. In certain embodiments, the
present invention provides a method for producing a therapeutic
composition comprising lipid encapsulated mRNA that encodes for a
protein that modulates a Nav1.5 channel in muscle tissue or in a
muscle cell.
[0242] In certain embodiments, the present invention provides a
method for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes a peptide or polypeptide for use in
the delivery to or treatment of the nervous system of a subject or
a nervous system cell. For example, in certain embodiments, the
present invention provides a method for producing a therapeutic
composition comprising lipid encapsulated mRNA that encodes for
survival motor neuron 1 protein. For example, in certain
embodiments, the present invention provides a method for producing
a therapeutic composition comprising lipid encapsulated mRNA that
encodes for survival motor neuron 2 protein. In certain
embodiments, the present invention provides a method for producing
a therapeutic composition comprising lipid encapsulated mRNA that
encodes for frataxin protein. In certain embodiments, the present
invention provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for ATP binding
cassette subfamily D member 1 (ABCD1) protein. In certain
embodiments, the present invention provides a method for producing
a therapeutic composition comprising lipid encapsulated mRNA that
encodes for CLN3 protein.
[0243] In certain embodiments, the present invention provides a
method for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes a peptide or polypeptide for use in
the delivery to or treatment of the blood or bone marrow of a
subject or a blood or bone marrow cell. In certain embodiments, the
present invention provides a method for producing a therapeutic
composition comprising lipid encapsulated mRNA that encodes for
beta globin protein. In certain embodiments, the present invention
provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for Bruton's
tyrosine kinase protein. In certain embodiments, the present
invention provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for one or clotting
enzymes, such as Factor VIII, Factor IX, Factor VII, and Factor
X.
[0244] In certain embodiments, the present invention provides a
method for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes a peptide or polypeptide for use in
the delivery to or treatment of the kidney of a subject or a kidney
cell. In certain embodiments, the present invention provides a
method for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes for collagen type IV alpha 5 chain
(COL4A5) protein.
[0245] In certain embodiments, the present invention provides a
method for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes a peptide or polypeptide for use in
the delivery to or treatment of the eye of a subject or an eye
cell. In certain embodiments, the present invention provides a
method for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes for ATP-binding cassette subfamily A
member 4 (ABCA4) protein. In certain embodiments, the present
invention provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for retinoschisin
protein. In certain embodiments, the present invention provides a
method for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes for retinal pigment
epithelium-specific 65 kDa (RPE65) protein. In certain embodiments,
the present invention provides a method for producing a therapeutic
composition comprising lipid encapsulated mRNA that encodes for
centrosomal protein of 290 kDa (CEP290).
[0246] In certain embodiments, the present invention provides a
method for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes a peptide or polypeptide for use in
the delivery of or treatment with a vaccine for a subject or a cell
of a subject. For example, in certain embodiments, the present
invention provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for an antigen from
an infectious agent, such as a virus. In certain embodiments, the
present invention provides a method for producing a therapeutic
composition comprising lipid encapsulated mRNA that encodes for an
antigen from influenza virus. In certain embodiments, the present
invention provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for an antigen from
respiratory syncytial virus. In certain embodiments, the present
invention provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for an antigen from
rabies virus. In certain embodiments, the present invention
provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for an antigen from
cytomegalovirus. In certain embodiments, the present invention
provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for an antigen from
rotavirus. In certain embodiments, the present invention provides a
method for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes for an antigen from a hepatitis
virus, such as hepatitis A virus, hepatitis B virus, or hepatis C
virus. In certain embodiments, the present invention provides a
method for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes for an antigen from human
papillomavirus. In certain embodiments, the present invention
provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for an antigen from
a herpes simplex virus, such as herpes simplex virus 1 or herpes
simplex virus 2. In certain embodiments, the present invention
provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for an antigen from
a human immunodeficiency virus, such as human immunodeficiency
virus type 1 or human immunodeficiency virus type 2. In certain
embodiments, the present invention provides a method for producing
a therapeutic composition comprising lipid encapsulated mRNA that
encodes for an antigen from a human metapneumovirus. In certain
embodiments, the present invention provides a method for producing
a therapeutic composition comprising lipid encapsulated mRNA that
encodes for an antigen from a human parainfluenza virus, such as
human parainfluenza virus type 1, human parainfluenza virus type 2,
or human parainfluenza virus type 3. In certain embodiments, the
present invention provides a method for producing a therapeutic
composition comprising lipid encapsulated mRNA that encodes for an
antigen from malaria virus. In certain embodiments, the present
invention provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for an antigen from
zika virus. In certain embodiments, the present invention provides
a method for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes for an antigen from chikungunya
virus.
[0247] In certain embodiments, the present invention provides a
method for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes for an antigen associated with a
cancer of a subject or identified from a cancer cell of a subject.
In certain embodiments, the present invention provides a method for
producing a therapeutic composition comprising lipid encapsulated
mRNA that encodes for an antigen determined from a subject's own
cancer cell, i.e., to provide a personalized cancer vaccine. In
certain embodiments, the present invention provides a method for
producing a therapeutic composition comprising lipid encapsulated
mRNA that encodes for an antigen expressed from a mutant KRAS
gene.
[0248] In certain embodiments, the present invention provides a
method for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes for an antibody. In certain
embodiments, the antibody can be a bi-specific antibody. In certain
embodiments, the antibody can be part of a fusion protein. In
certain embodiments, the present invention provides a method for
producing a therapeutic composition comprising lipid encapsulated
mRNA that encodes for an antibody to OX40. In certain embodiments,
the present invention provides a method for producing a therapeutic
composition comprising lipid encapsulated mRNA that encodes for an
antibody to VEGF. In certain embodiments, the present invention
provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for an antibody to
tissue necrosis factor alpha. In certain embodiments, the present
invention provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for an antibody to
CD3. In certain embodiments, the present invention provides a
method for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes for an antibody to CD19.
[0249] In certain embodiments, the present invention provides a
method for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes for an immunomodulator. In certain
embodiments, the present invention provides a method for producing
a therapeutic composition comprising lipid encapsulated mRNA that
encodes for Interleukin 12. In certain embodiments, the present
invention provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for Interleukin 23.
In certain embodiments, the present invention provides a method for
producing a therapeutic composition comprising lipid encapsulated
mRNA that encodes for Interleukin 36 gamma. In certain embodiments,
the present invention provides a method for producing a therapeutic
composition comprising lipid encapsulated mRNA that encodes for a
constitutively active variant of one or more stimulator of
interferon genes (STING) proteins.
[0250] In certain embodiments, the present invention provides a
method for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes for an endonuclease. In certain
embodiments, the present invention provides a method for producing
a therapeutic composition comprising lipid encapsulated mRNA that
encodes for an RNA-guided DNA endonuclease protein, such as Cas 9
protein. In certain embodiments, the present invention provides a
method for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes for a meganuclease protein. In
certain embodiments, the present invention provides a method for
producing a therapeutic composition comprising lipid encapsulated
mRNA that encodes for a transcription activator-like effector
nuclease protein. In certain embodiments, the present invention
provides a method for producing a therapeutic composition
comprising lipid encapsulated mRNA that encodes for a zinc finger
nuclease protein.
[0251] In certain embodiments, the present invention provides a
method for producing a therapeutic composition comprising lipid
encapsulated mRNA that encodes for treating an ocular disease. In
some embodiments, the method is used for producing a therapeutic
composition comprising lipid encapsulated mRNA encoding
retinoschisin.
EXAMPLES
[0252] The following examples, including the experiments conducted
and results achieved are provided for illustrative purposes only
and are not to be construed as limiting upon the present
disclosure.
Lipid Materials
[0253] The formulations described in the following Examples, unless
otherwise specified, contain a multi-component lipid mixture of
varying ratios employing one or more cationic lipids, helper lipids
(e.g. non-cationic lipids and/or cholesterol lipids), and PEGylated
lipids designed to encapsulate various nucleic acid materials.
Example 1: Gravity-Based Nucleic Acid Encapsulation
[0254] This example illustrates a gravity-based nucleic acid
encapsulation process. As used herein, Process A refers to a
conventional method of encapsulating mRNA by mixing mRNA with a
mixture of lipids, without first pre-forming the lipids into lipid
nanoparticles. As used herein, Process B refers to a process of
encapsulating messenger RNA (mRNA) by mixing pre-formed lipid
nanoparticles with mRNA. As compared to Process B, Process A does
not involve pre-formation of lipid nanoparticles. Process A and
Process B include those described in WO2016004318 and WO2018089801,
respectively, which are hereby incorporated by reference.
[0255] FIG. 1 illustrates an exemplary encapsulation process using
the methods described herein. The exemplary encapsulation process
of the present invention can be applied to both Process A and
Process B. The exemplary process shown in FIG. 1 includes 1) a
first reservoir to provide a desired nucleic acid in aqueous
solution; 2) a second reservoir to provide a solution of lipids
and/or lipid nanoparticles (LNPs); 3) conduits for the first and
second reservoirs to allow for flow of nucleic acids, and lipids
and/or LNPs; 4) a junction for mixing the nucleic acids and lipids
and/or LNPs; and 5) a receptacle or conduit for collecting the
mixed/encapsulated nucleic acids in LNPs.
[0256] The process uses gravity and atmospheric pressure as forces
to drive the flow of liquid from Reservoir 1 (mRNA solution
containing reservoir), and Reservoir 2 (lipids or LNP solution
containing reservoir) through Conduit 1 (mRNA solution flow) and
through Conduit 2 (lipid or LNP solution flow), respectively. The
solutions from Reservoir 1 and Reservoir 2 meet at the junction
(i.e. a "Y" connector or a "T" connector) and are thus mixed
together at a specific flow rate. The mixing of the mRNA solution
and the lipid or LNP containing solution results in the
encapsulation of the mRNA in a lipid nanoparticle. The encapsulated
mRNA is subsequently collected in a receptacle.
[0257] Various junctions can be used with the process disclosed
herein. As illustrated in FIGS. 2A and 2B, respectively, options of
junctions include, for example, use of a "T" connector or a "Y"
connector.
[0258] This process was used to successfully encapsulate mRNA using
various cationic lipids, including ones listed in Table 1, applying
to both Process A and Process B. The measured atmospheric pressure
and head pressure were essentially 0. The results of the
encapsulation processes runs are shown in Table 1 below.
TABLE-US-00001 TABLE 1 Encapsulation of mRNA using a Gravity- Based
Encapsulation Process % mRNA Cationic lipid Size (nm) encapsulation
PDI cKK-E12 (1) 99 79 MC3 (1) 89 83 CCBene (1) 102 87 cKK-E12 (2)
72 0.218 OF-02/cKK- 82 0.126 E18:2/ML7 cDD-TE-E12 93 0.259 MC3 (2)
68 0.156 CCBene (2) 69 0.112 RL2-DMP-07D 84 0.147 ICE 49 0.283
cKK-E12 (Process B) 92 0.133 * Process A was applied unless
indicated otherwise.
[0259] As is shown in Table 1, the gravity encapsulation process
resulted in encapsulation efficiencies of between about 79 and 87%
for encapsulation with cKK-E12 and CCBene, respectively. Moreover,
the sizes of the encapsulated mRNA ranged from about 49 nm to about
102 nm, with low PDI values, all below 0.3.
Example 2: Control of Liquid Flow Rate in the Process
[0260] The liquid flow rate can be controlled in the process shown
in FIG. 1 in order to achieve a desired flow rate and resultant
mixing properties. One way to control the flow rates and the mixing
of the solutions contained in the reservoirs is to adjust the
diameter of at least one of the Reservoir (e.g. Reservoir 1 and/or
Reservoir 2), the conduit, and/or the junction. FIGS. 3A and 3B
illustrate adjustments to the diameter of a conduit and the
resultant impact had on the liquid flow rate due to conduit
diameter. As can be seen in FIGS. 3A and 3B, the larger the
diameter, the greater the flow of liquid through the conduit.
[0261] One manner to achieve a desired diameter and resultant flow
rate is by placing a constrictor (e.g., pinch bulb, lid, and clip)
in one or more of the reservoir, conduit and/or junction. Placement
of constrictors at each of these parts of the process is
illustrated in FIG. 4A-4D. The placement of the constrictor will
alter the diameter through which liquid flows from the reservoir,
thus leading to adjustments in the flow rate and the mixing that
occurs at the junction.
[0262] Another manner to control the flow rate and the resultant
mixing process is to move the connector upwards, such that the
conduit lines are allowed to purge-fill, and at the same time
restricting liquid flow due to height (i.e. gravity control). The
liquids from Reservoir 1 and Reservoir 2 will mix upon moving the
connector downwards. This manner of controlling the mixing process
is illustrated in FIGS. 5A and 5B. Alternatively, the system can be
held constant, adding liquids so that the formulation mix is the
result of the fixed system. This is shown in FIG. 6.
Example 3: High Throughput Formulations Processes
[0263] The process as described herein can also be used in
high-throughput scenarios. For example, a series of processes as
described herein can be connected such that the process would
comprise at least 10, 20, 30, 40, 50, 100, 150, 200, 250, 300 or
more pairs of first conduit streams and second conduit streams.
This would allow, for example, that the first conduit stream
provide multiple mRNA solutions if so desired. Likewise, this the
second conduit stream can provide multiple lipid solutions if so
desired. In this manner, an assembly-line like approach is
achieved, such that liquids are added to pairs of reservoirs at the
same time, then the liquids are added to the next pairs of
reservoirs in succession. This is illustrated in FIG. 7. An
exemplary high throughput process is shown in FIG. 8.
EQUIVALENTS AND SCOPE
[0264] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. The scope of the present invention is not intended to be
limited to the above Description, but rather is as set forth in the
following claims:
* * * * *